COMPLEMENT COMPONENT iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The invention relates to iRNA, e.g., double-stranded ribonucleic acid (dsRNA), compositions targeting the complement factor B (CFB) gene, the complement component C3 gene, and the complement component C9 gene and methods of using such iRNA, e.g., dsRNA, compositions to inhibit expression of CFB, C9 and/or C3 and to treat subjects having a complement component-associated disease, e.g., paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/925,463, filed on Jul. 10, 2020, which is a divisional application ofU.S. application Ser. No. 16/574,158, filed on Sep. 18, 2019, which is adivisional of U.S. patent application Ser. No. 15/176,231, filed on Jun.8, 2016, now U.S. Pat. No. 10,465,194, issued on Nov. 5, 2019, which isa 35 § U.S.C. 111(a) continuation application which claims the benefitof priority to PCT/US2014/069951, filed on Dec. 12, 2014, and to U.S.Provisional Patent Application Ser. No. 61/915,210, filed on Dec. 12,2013. The entire contents of each of the foregoing applications areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 4, 2021, isnamed 121301_01105_SL.txt and is 266,238 bytes in size.

BACKGROUND OF THE INVENTION

Complement was first discovered in the 1890s when it was found to aid or“complement” the killing of bacteria by heat-stable antibodies presentin normal serum (Walport, M. J. (2001) N Engl J Med. 344:1058). Thecomplement system consists of more than 30 proteins that are eitherpresent as soluble proteins in the blood or are present asmembrane-associated proteins. Activation of complement leads to asequential cascade of enzymatic reactions, known as complementactivation pathways resulting in the formation of the potentanaphylatoxins C3a and C5a that elicit a plethora of physiologicalresponses that range from chemoattraction to apoptosis. Initially,complement was thought to play a major role in innate immunity where arobust and rapid response is mounted against invading pathogens.However, recently it is becoming increasingly evident that complementalso plays an important role in adaptive immunity involving T and Bcells that help in elimination of pathogens (Dunkelberger J R and Song WC. (2010) Cell Res. 20:34; Molina H, et al. (1996) Proc Natl Acad SciUSA. 93:3357), in maintaining immunologic memory preventing pathogenicre-invasion, and is involved in numerous human pathological states (Qu,H, et al. (2009) Mol Immunol. 47:185; Wagner, E. and Frank M M. (2010)Nat Rev Drug Discov. 9:43).

Complement activation is known to occur through three differentpathways: alternate, classical and lectin (FIG. 1) involving proteinsthat mostly exist as inactive zymogens that are then sequentiallycleaved and activated.

The classical pathway is often activated by antibody-antigen complexesor by the C-reactive protein (CRP), both of which interact withcomplement component C1q. In addition, the classical pathway can beactivated by phosphatidyl serine present in apoptotic bodies in theabsence of immune complexes.

The lectin pathway is initiated by the mannose-binding lectins (MBL)that bind to complex carbohydrate residues on the surface of pathogens.The activation of the classical pathway or the lectin pathway leads toactivation of the (C4b2b) C3 convertase.

The alternate pathway is activated by the binding of C3b, which isspontaneously generated by the hydrolysis of C3, on targeted surfaces.This surface-bound C3b is then recognized by factor B, forming thecomplex C3bB. The C3bB complex, in turn, is cleaved by factor D to yieldthe active form of the C3 convertase of the AP (C3bBb). Both types of C3convertases will cleave C3, forming C3b. C3b then either binds to morefactor B, enhancing the complement activation through the AP (theso-called alternative or amplification loop), or leads to the formationof the active C5 convertase (C3bBbC3b or C4bC2bC3b), which cleaves C5and triggers the late events that result in the formation of themembrane attack complex (MAC) (C5b-9).

Inappropriate activation of the complement system is responsible forpropagating and/or initiating pathology in many different diseases,including, for example, paroxysmal nocturnal hemoglobinuria, atypicalhemolytic uremic syndrome, rheumatoid arthritis, ischemia-reperfusioninjuries and neurodegenerative diseases.

To date, only one therapeutic that targets the C5-05a axis is availablefor the treatment of complement component-associated diseases, theanti-05 antibody, eculizumab (Soliris®). Although eculizumab has beenshown to be effective for the treatment of paroxysmal nocturnalhemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) andis currently being evaluated in clinical trials for additionalcomplement component-associated diseases, eculizumab therapy requiresweekly high dose infusions followed by biweekly maintenance infusions ata high cost. Furthermore, approximately 50% of eculizumab-treated PNHsubjects have low level of hemolysis and require residual transfusions(Hill A, et al. (2010) Haematologica 95(4):567-73). Accordingly, thereis a need in the art for alternative therapies and combination therapiesfor subjects having a complement component-associated disease.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a CFB gene. The CFB gene may be within a cell, e.g., acell within a subject, such as a human.

The present invention also provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a C3 gene. The C3 gene may be within a cell, e.g., a cellwithin a subject, such as a human.

In addition, the present invention provides iRNA compositions whicheffect the RNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a C9 gene. The C9 gene may be within a cell, e.g., a cellwithin a subject, such as a human.

The present invention also provides methods and combination therapiesfor treating a subject having a disorder that would benefit frominhibiting or reducing the expression of a CFB, C3, and/or C9 gene,e.g., a complement component-associated disease, such as paroxysmalnocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome(aHUS) using iRNA compositions which effect the RNA-induced silencingcomplex (RISC)-mediated cleavage of RNA transcripts of a CFB, C3, and/orC9 gene for inhibiting the expression of a CFB, C3, and/or C9 gene.

Accordingly, in one aspect the present invention providesdouble-stranded ribonucleic acids (dsRNA) for inhibiting expression ofcomplement factor B (CFB) in a cell, wherein the dsRNA comprises a sensestrand and an antisense strand, wherein the sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom any one of the nucleotide sequence of SEQ ID NOs:1-5, 27, and 30,and the antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the nucleotidesequence of SEQ ID NOs:12-16, 33, and 36.

In another aspect the present invention provides double-strandedribonucleic acids (dsRNA) for inhibiting expression of complement factorB (CFB) in a cell, wherein the dsRNA comprises a sense strand and anantisense strand, the antisense strand comprising a region ofcomplementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the antisensesequences listed in Table 3 and 4.

In one embodiment, the sense and antisense strands comprise sequencesselected from the group consisting of AD-60304, AD-60331, and AD-60344and any one of the agents listed in Tables 3 and 4.

In one embodiment the region of complementarity consists of thenucleotide sequence of one of the antisense sequences of any one ofTables 3 and 4.

In one embodiment, the dsRNA comprises a sense strand consisting of thenucleotide sequence of a sense strand sequence selected from thesequence of any one of Tables 3 and 4, and an antisense strandconsisting of the nucleotide sequence of an antisense sequence selectedfrom the sequences of any one of Tables 3 and 4. In another aspect thepresent invention provides double-stranded ribonucleic acids (dsRNA) forinhibiting expression of complement component C3 in a cell, wherein thedsRNA comprises a sense strand and an antisense strand, wherein thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from any one of the nucleotide sequence ofSEQ ID NOs:6-8, 28, and 31, and the antisense strand comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromany one of the nucleotide sequence of SEQ ID NOs:17-19, 34, and 37.

In another aspect the present invention provides double-strandedribonucleic acids (dsRNA) for inhibiting expression of complementcomponent C3 in a cell, wherein the dsRNA comprises a sense strand andan antisense strand, the antisense strand comprising a region ofcomplementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the antisensesequences listed in Table 5 and 6.

In one embodiment, the sense and antisense strands comprise sequencesselected from the group consisting of AD-60169 and any one of the agentslisted in Tables 5 and 6.

In one embodiment the region of complementarity consists of thenucleotide sequence of one of the antisense sequences of any one ofTables 5 and 6.

In one embodiment, the dsRNA comprises a sense strand consisting of thenucleotide sequence of a sense strand sequence selected from thesequence of any one of Tables 5 and 6, and an antisense strandconsisting of the nucleotide sequence of an antisense sequence selectedfrom the sequences of any one of Tables 5 and 6.

In another aspect the present invention provides double-strandedribonucleic acids (dsRNA) for inhibiting expression of complementcomponent C9 in a cell, wherein the dsRNA comprises a sense strand andan antisense strand, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequence of SEQ ID NOs:9-11, 29, and 32, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from any one of the nucleotide sequence ofSEQ ID NOs:20-22, 35, and 38.

In another aspect the present invention provides double-strandedribonucleic acids (dsRNA) for inhibiting expression of complementcomponent C9 in a cell, wherein the dsRNA comprises a sense strand andan antisense strand, the antisense strand comprising a region ofcomplementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the antisensesequences listed in Table 7 and 8.

In one embodiment, the sense and antisense strands comprise sequencesselected from the group consisting of any one of the agents listed inTables 7 and 8.

In one embodiment the region of complementarity consists of thenucleotide sequence of one of the antisense sequences of any one ofTables 7 and 8.

In one embodiment, the dsRNA comprises a sense strand consisting of thenucleotide sequence of a sense strand sequence selected from thesequence of any one of Tables 7 and 8, and an antisense strandconsisting of the nucleotide sequence of an antisense sequence selectedfrom the sequences of any one of Tables 7 and 8.

The dsRNA may include at least one modified nucleotide, e.g., a2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, a deoxynucleotide, a 3′-terminaldeoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, aterminal nucleotide linked to a cholesteryl derivative or a dodecanoicacid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, alocked nucleotide, an unlocked nucleotide, a conformationally restrictednucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide,2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, a non-natural base comprisingnucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitolmodified nucleotide, a cyclohexenyl modified nucleotide, a nucleotidecomprising a phosphorothioate group, a nucleotide comprising amethylphosphonate group, a nucleotide comprising a 5′-phosphate, and anucleotide comprising a 5′-phosphate mimic.

In one embodiment, substantially all the nucleotides of the sense strandand the antisense strand are modified nucleotides. In anotherembodiment, all the nucleotides of the sense strand and the antisensestrand are modified nucleotides.

The region of complementarity may be at least 17 nucleotides in length,such as 19 nucleotides in length, or no more than 30 nucleotides inlength.

The region of complementarity may be between 19 and 21 nucleotides inlength.

At least one strand of the dsRNA may include a 3′ overhang of at least 1nucleotide, or at least 2 nucleotides.

The dsRNA may further include a ligand. In one embodiment, the ligand isconjugated to the 3′ end of the sense strand of the dsRNA. In oneembodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.In one embodiment, the ligand is

In one embodiment, the dsRNA is conjugated to the ligand as shown in thefollowing schematic

and, wherein X is O or S.

In one embodiment, the X is O.

In another aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of complement factor B (CFB)in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding CFB, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of complement component 3(C3) in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding C3, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In a further aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of complement component 9(C9) in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding C9, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0;or both i and j are 1.

In one embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0;or both k and l are 1.

In one embodiment, XXX is complementary to X′X′X′, YYY is complementaryto Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand.

In one embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13positions of the antisense strand from the 5′-end.

In one embodiment, the Y′ is 2′-O-methyl.

In one embodiment, formula (III) is represented by formula (IIIa):

sense: 5′ n_(p)-N_(a)—YYY—N_(a)-n_(q) 3′

antisense: 3′ n_(p′)-N_(a′)—Y′Y′Y′—N_(a′)-n_(q′) 5′  (IIIa).

In one embodiment, formula (III) is represented by formula (IIIb):

sense: 5′ n_(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n_(q) 3′

antisense: 3′ n_(p′)-N_(a′)—Y′Y′Y′—N_(b′)—Z′Z′Z′—N_(a′)-n_(q′)5′  (IIIb)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In one embodiment, formula (III) is represented by formula (IIIc):

sense: 5′ n_(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n_(q) 3′

antisense: 3′ n_(p′)-N_(a′)—X′X′X′—N_(b)′—Y′Y′Y′—N_(a′)-n_(q′) 5′ (IIIc)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In one embodiment, formula (III) is represented by formula (IIId):

sense: 5′ n_(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n_(q) 3′

antisense: 3′n_(p′)-N_(a′)—X′X′X′—N_(b′)—Y′Y′Y′—N_(b′)—Z′Z′Z′—N_(a′)-n_(q′) 5′  (IIId)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.

The double-stranded region may 15-30 nucleotide pairs in length, 17-23nucleotide pairs in length, 17-25 nucleotide pairs in length, 23-27nucleotide pairs in length, 19-21 nucleotide pairs in length, or 21-23nucleotide pairs in length.

In one embodiment, each strand has 15-30 nucleotides.

In one embodiment, the modifications on the nucleotides are selectedfrom the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl,and combinations thereof.

In one embodiment, the modifications on the nucleotides are 2′-O-methylor 2′-fluoro modifications.

In one embodiment, the ligand is one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In one embodiment, the ligand is

In one embodiment, the ligand is attached to the 3′ end of the sensestrand.

In one embodiment, the RNAi agent is conjugated to the ligand as shownin the following schematic

and, wherein X is O or S.

In one embodiment, the X is O.

In one embodiment, the agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand. In oneembodiment, the strand is the antisense strand. In another embodiment,the strand is the sense strand.

In another embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand. In oneembodiment, the strand is the antisense strand. In another embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. In one embodiment, the strand is the antisense strand.

In one embodiment, the base pair at the 1 position of the 5′-end of theantisense strand of the duplex is an AU base pair.

In one embodiment, the Y nucleotides contain a 2′-fluoro modification.

In one embodiment, the Y′ nucleotides contain a 2′-O-methylmodification.

In one embodiment, p′>0. In another embodiment, p′=2.

In one embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides arecomplementary to the target mRNA.

In another embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides arenon-complementary to the target mRNA.

In one embodiment, the sense strand has a total of 21 nucleotides andthe antisense strand has a total of 23 nucleotides.

In one embodiment, at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage. In another embodiment, alln_(p)′ are linked to neighboring nucleotides via phosphorothioatelinkages.

In one embodiment, the RNAi agent is selected from the group of RNAiagents listed in Tables 3 and 4. In one embodiment, the RNAi agent isselected from the group of RNAi agents AD-60304, AD-60331, and AD-60344.

In another embodiment, the RNAi agent is selected from the group of RNAiagents listed in Tables 5 and 6.

In yet another embodiment, the RNAi agent is selected from the group ofRNAi agents listed in Tables 7 and 8.

In one aspect, the present invention provides double stranded RNAiagents comprising the RNAi agents listed in any one of Tables 3, 5, and7.

In one aspect, the present invention provides compositions comprising amodified antisense polynucleotide agent. The agents are capable ofinhibiting the expression of Complement Factor B (CFB) in a cell, andinclude a sequence complementary to a sense sequence selected from thegroup of the sequences listed in Table 3, wherein the polynucleotide isabout 14 to about 30 nucleotides in length.

In another aspect, the present invention provides compositionscomprising a modified antisense polynucleotide agent. The agents arecapable of inhibiting the expression of Complement Component 3 (C3) in acell, and include a sequence complementary to a sense sequence selectedfrom the group of the sequences listed in Table 5, wherein thepolynucleotide is about 14 to about 30 nucleotides in length.

In yet another aspect, the present invention provides compositionscomprising a modified antisense polynucleotide agent. The agents arecapable of inhibiting the expression of Complement Component 9 (C9) in acell, and include a sequence complementary to a sense sequence selectedfrom the group of the sequences listed in Table 7, wherein thepolynucleotide is about 14 to about 30 nucleotides in length.

In one aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of Complement Factor B (CFB)in a cell. The agent include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding CFB, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In another aspect, the invention provides double stranded RNAi agentscapable of inhibiting the expression of Complement Factor B (CFB) in acell. The agents include a sense strand complementary to an antisensestrand, wherein the antisense strand comprises a region complementary topart of an mRNA encoding CFB, wherein each strand is about 14 to about30 nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of Complement Factor B (CFB)in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding CFB, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In yet a further aspect, the present invention provides double strandedRNAi agents capable of inhibiting the expression of Complement Factor B(CFB) in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding CFB, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In another aspect, the invention provides double stranded RNAi agentscapable of inhibiting the expression of Complement Factor B (CFB) in acell. The agents include a sense strand complementary to an antisensestrand, wherein the antisense strand comprises a region complementary topart of an mRNA encoding CFB, wherein each strand is about 14 to about30 nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′ n_(p)-N_(a)—YYY—N_(a)-n_(q) 3′

antisense: 3′ n_(p)′-N_(a)′—Y′Y′Y′—N_(a)′-n_(q)′ 5′  (IIIa)

wherein:

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides, and wherein themodifications are 2′-O-methyl or 2′-fluoro modifications;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In one aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of Complement Component 3(C3) in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding C3, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of Complement Component 3(C3) in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding C3, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In another aspect, the invention provides double stranded RNAi agentscapable of inhibiting the expression of Complement Component 3 (C3) in acell. The agents include a sense strand complementary to an antisensestrand, wherein the antisense strand comprises a region complementary topart of an mRNA encoding C3, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In yet another aspect, the present invention provides double strandedRNAi agents capable of inhibiting the expression of Complement Component3 (C3) in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding C3, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

-   -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides, and wherein the modifications are        2′-O-methyl or 2′-fluoro modifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In one aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of Complement Component 3(C3) in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding C3, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—YYY—N_(a)-n_(q) 3′

antisense: 3′ n_(p)′-N_(a)′—Y′Y′Y′—N_(a)′-n_(q)′ 5′  (IIIa)

wherein:

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

-   -   YYY and Y′Y′Y′ each independently represent one motif of three        identical modifications on three consecutive nucleotides, and        wherein the modifications are 2′-O-methyl or 2′-fluoro        modifications;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In another aspect, the present invention provides double stranded RNAiagents capable of inhibiting the expression of Complement Component 9(C9) in a cell. The agents include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding C9, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In one aspect, the invention provides double stranded RNAi agentscapable of inhibiting the expression of Complement Component 9 (C9) in acell. The agents include a sense strand complementary to an antisensestrand, wherein the antisense strand comprises a region complementary topart of an mRNA encoding C9, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In another aspect, the invention provides double stranded RNAi agentscapable of inhibiting the expression of Complement Component 9 (C9) in acell. The agents include a sense strand complementary to an antisensestrand, wherein the antisense strand comprises a region complementary topart of an mRNA encoding C9, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In another aspect, the invention provides double stranded RNAi agentscapable of inhibiting the expression of Complement Component 9 (C9) in acell. The agents include a sense strand complementary to an antisensestrand, wherein the antisense strand comprises a region complementary topart of an mRNA encoding C9, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In a further aspect, the present invention provisos double stranded RNAiagents capable of inhibiting the expression of Complement Component 9(C9) in a cell. The agent include a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding C9, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)—YYY—N_(a)-n_(q) 3′

antisense: 3′ n_(p)′-N_(a)′—Y′Y′Y′—N_(a)′-n_(q)′ 5′  (IIIa)

wherein:

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides, and wherein themodifications are 2′-O-methyl or 2′-fluoro modifications;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In another aspect, the invention provides double stranded RNAi agentsfor inhibiting expression of complement factor B (CFB) in a cell,wherein the double stranded RNAi agent comprises a sense strand and anantisense strand forming a double stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of any one of SEQ IDNOs:1-5, 27, and 30, and the antisense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequence of SEQ ID NOs:12-16, 33, and 36, whereinsubstantially all of the nucleotides of the sense strand comprise amodification selected from the group consisting of a 2′-O-methylmodification and a 2′-fluoro modification, wherein the sense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus, wherein substantially all of the nucleotides of theantisense strand comprise a modification selected from the groupconsisting of a 2′-O-methyl modification and a 2′-fluoro modification,wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and wherein the sensestrand is conjugated to one or more GalNAc derivatives attached througha branched bivalent or trivalent linker at the 3′-terminus.

In another aspect, the present invention provides double stranded RNAiagents for inhibiting expression of complement component C3 in a cell,wherein the double stranded RNAi agent comprises a sense strand and anantisense strand forming a double stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of any one of SEQ IDNOs:6-8, 28, and 31, and the antisense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequence of SEQ ID NOs:17-19, 34, and 37, whereinsubstantially all of the nucleotides of the sense strand comprise amodification selected from the group consisting of a 2′-O-methylmodification and a 2′-fluoro modification, wherein the sense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus, wherein substantially all of the nucleotides of theantisense strand comprise a modification selected from the groupconsisting of a 2′-O-methyl modification and a 2′-fluoro modification,wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and wherein the sensestrand is conjugated to one or more GalNAc derivatives attached througha branched bivalent or trivalent linker at the 3′-terminus.

In yet another aspect, the present invention provides double strandedRNAi agents for inhibiting expression of complement component C9 in acell, wherein the double stranded RNAi agent comprises a sense strandand an antisense strand forming a double stranded region, wherein thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of any one ofSEQ ID NOs:9-11, 29, and 32, and the antisense strand comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromany one of the nucleotide sequence of SEQ ID NOs:20-22, 35, and 38,wherein substantially all of the nucleotides of the sense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification, wherein the sensestrand comprises two phosphorothioate internucleotide linkages at the5′-terminus, wherein substantially all of the nucleotides of theantisense strand comprise a modification selected from the groupconsisting of a 2′-O-methyl modification and a 2′-fluoro modification,wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and wherein the sensestrand is conjugated to one or more GalNAc derivatives attached througha branched bivalent or trivalent linker at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In another aspect, the present invention provides cells containing theagents of the invention.

In one aspect, the invention provides vectors encoding at least onestrand of the agents of the invention.

In another aspect, the invention provides cells comprising the vectorsof the invention.

In one aspect, the present invention provides pharmaceuticalcompositions for inhibiting expression of a complement component factorB gene comprising the agents the invention.

In another aspect, the present invention provides pharmaceuticalcompositions for inhibiting expression of a complement component C3 genecomprising the agents of the invention.

In yet another aspect, the present invention provides pharmaceuticalcompositions for inhibiting expression of a complement component C9 genecomprising the agents of the invention.

In one embodiment, the RNAi agent is administered in an unbufferedsolution.

In one embodiment, the unbuffered solution is saline or water.

In one embodiment, the RNAi agent is administered with a buffersolution.

In one embodiment, the buffer solution comprises acetate, citrate,prolamine, carbonate, or phosphate or any combination thereof.

In one embodiment, the buffer solution is phosphate buffered saline(PBS).

In one aspect, the present invention provides methods of inhibitingcomplement factor B (CFB) expression in a cell. The methods includecontacting the cell with the agent of a the invention or apharmaceutical composition of the invention, and maintaining the cellproduced for a time sufficient to obtain degradation of the mRNAtranscript of a CFB gene, thereby inhibiting expression of the CFB genein the cell.

In another aspect, the present invention provides methods of inhibitingcomplement component 3 (C3) expression in a cell. The methods includecontacting the cell with the agent of a the invention or apharmaceutical composition of the invention, and maintaining the cellproduced for a time sufficient to obtain degradation of the mRNAtranscript of a C3 gene, thereby inhibiting expression of the C3 gene inthe cell.

In yet another aspect, the present invention provides methods ofinhibiting complement component 9 (C9) expression in a cell. The methodsinclude contacting the cell with the agent of a the invention or apharmaceutical composition of the invention, and maintaining the cellproduced for a time sufficient to obtain degradation of the mRNAtranscript of a C9 gene, thereby inhibiting expression of the C9 gene inthe cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human.

In one embodiment, the human subject suffers from a complementcomponent-associated disease.

In one embodiment, the complement component-associated disease isselected from the group consisting of paroxysmal nocturnalhemoglobinuria (PNH), asthma, rheumatoid arthritis, systemic lupuserythmatosis, glomerulonephritis, psoriasis, dermatomyositis bullouspemphigoid, atypical hemolytic uremic syndrome, Shiga toxin E.coli-related hemolytic uremic syndrome, myasthenia gravis,neuromyelistis optica, dense deposit disease, C3 neuropathy, age-relatedmacular degeneration, cold agglutinin disease, anti-neutrophilcytoplasmic antibody-associated vasculitis, humoral and vasculartransplant rejection, graft dysfunction, myocardial infarction, asensitized recipient of a transplant, and sepsis.

In one embodiment, the complement component-associated disease isparoxysmal nocturnal hemoglobinuria (PNH).

In another embodiment, the complement component-associated disease isatypical hemolytic uremic syndrome (aHUS).

In one embodiment, the CFB expression is inhibited by at least about30%.

In one embodiment, the C3 expression is inhibited by at least about 30%.

In one embodiment, the C9 expression is inhibited by at least about 30%.

In one embodiment, the agent is administered at a dose of about 0.01mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

In another embodiment, the agent is administered at a dose of about 10mg/kg to about 30 mg/kg.

In one embodiment, the agent is administered subcutaneously.

In another embodiment, the agent is administered intravenously.

In one aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction incomplement factor B (CFB) expression. The methods include administeringto the subject a therapeutically effective amount of the agent of theinvention, thereby treating the subject.

In another aspect, the present invention provides methods of preventingat least one symptom in a subject having a disease or disorder thatwould benefit from reduction in complement factor B (CFB) expression.The methods include administering to the subject a therapeuticallyeffective amount of the agent of the invention, thereby preventing atleast one symptom in the subject having a disorder that would benefitfrom reduction in CFB expression.

In yet another aspect, the present invention provides methods oftreating a subject having a disorder that would benefit from reductionin complement component C3 (C3) expression. The methods includeadministering to the subject a therapeutically effective amount of theagent of the invention, thereby treating the subject.

In one aspect, the present invention provides methods of preventing atleast one symptom in a subject having a disease or disorder that wouldbenefit from reduction in complement component C3 (C3) expression. Themethods include administering to the subject a therapeutically effectiveamount of the agent of the invention, thereby preventing at least onesymptom in the subject having a disorder that would benefit fromreduction in C3 expression.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction incomplement component C9 (C9) expression. The methods includeadministering to the subject a therapeutically effective amount of theagent of the invention, thereby treating the subject.

In one aspect, the present invention provides methods of preventing atleast one symptom in a subject having a disease or disorder that wouldbenefit from reduction in complement component C9 (C9) expression. Themethods include administering to the subject a therapeutically effectiveamount of the agent of the invention, thereby preventing at least onesymptom in the subject having a disorder that would benefit fromreduction in C9 expression.

In one embodiment, the disorder is a complement component-associateddisease.

In one embodiment, the complement component-associated disease isselected from the group consisting of paroxysmal nocturnalhemoglobinuria (PNH), asthma, rheumatoid arthritis, systemic lupuserythmatosis, glomerulonephritis, psoriasis, dermatomyositis bullouspemphigoid, atypical hemolytic uremic syndrome, Shiga toxin E.coli-related hemolytic uremic syndrome, myasthenia gravis,neuromyelistis optica, dense deposit disease, C3 neuropathy, age-relatedmacular degeneration, cold agglutinin disease, anti-neutrophilcytoplasmic antibody-associated vasculitis, humoral and vasculartransplant rejection, graft dysfunction, myocardial infarction, asensitized recipient of a transplant, and sepsis.

In one embodiment, the complement component-associated disease isparoxysmal nocturnal hemoglobinuria (PNH).

In another embodiment, the complement component-associated disease isatypical hemolytic uremic syndrome (aHUS).

In one embodiment, the administration of the agent to the subject causesa decrease in hemolysis and/or a decrease in CFB protein accumulation.

In one embodiment, the administration of the agent to the subject causesa decrease in hemolysis and/or a decrease in C3 protein accumulation.

In one embodiment, the administration of the agent to the subject causesa decrease in hemolysis and/or a decrease in C9 protein accumulation.

In one embodiment, the methods further include administration ofeculizumab to the subject.

In another embodiment, the methods further include administration ofcompstatin to the subject.

In one embodiment, the agent is administered at a dose of about 0.01mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

In another embodiment, the agent is administered at a dose of about 10mg/kg to about 30 mg/kg.

In yet another embodiment, the agent is administered at a dose selectedfrom the group consisting of 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 10mg/kg, and 30 mg/kg.

In one embodiment, the agent is administered to the subject once a week.

In another embodiment, the agent is administered to the subject twice amonth.

In one embodiment, the methods further include measuring LDH levels inthe subject.

In one aspect, the present invention provides methods of inhibiting theexpression of complement factor B (CFB) in a subject. The methodsinclude administering to the subject a therapeutically effective amountof the agent of the invention, thereby inhibiting the expression of CFBin the subject.

In another aspect, the present invention provides methods of inhibitingthe expression of complement component C3 (C3) in a subject. The methodsinclude administering to the subject a therapeutically effective amountof the agent of the invention, thereby inhibiting the expression of C3in the subject.

In yet another aspect, the present invention provides methods ofinhibiting the expression of complement component C9 (C9) in a subject.The methods include administering to the subject a therapeuticallyeffective amount of the agent of any one of the invention, therebyinhibiting the expression of C9 in the subject.

In one embodiment, the methods further include administering eculizumabto the subject.

In another embodiment, the methods further include administeringcompstatin to the subject.

In one embodiment, the agent is administered at a dose of about 0.01mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

In another embodiment, the agent is administered at a dose of about 10mg/kg to about 30 mg/kg.

In yet another embodiment, the agent is administered at a dose selectedfrom the group consisting of 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg.

In one embodiment, the agent is administered to the subject once a week.

In another embodiment, the dsRNA agent is administered to the subjecttwice a month.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the three complement pathways: alternative,classical and lectin.

FIG. 2 is a graph showing the percentage of complement factor B (CFB)mRNA remaining in C57BL/6 mice 96 hours after a single 1 mg/kg or 10mg/kg dose of the indicated iRNAs.

FIG. 3 is a graph showing the percentage of complement factor B (CFB)mRNA remaining in C57BL/6 mice 72 hours after a single 1.25 mg/kg, 2.5mg/kg, or 10 mg/kg dose of AD-60331.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a complement component gene, i.e., a CFB, C3, or C9 gene.The gene may be within a cell, e.g., a cell within a subject, such as ahuman.

The present invention also provides methods and combination therapiesfor treating a subject having a disorder that would benefit frominhibiting or reducing the expression of a CFB, C9, and/or C3 gene,e.g., a complement component-associated disease, such as paroxysmalnocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome(aHUS) using iRNA compositions which effect the RNA-induced silencingcomplex (RISC)-mediated cleavage of RNA transcripts of a CFB, C3, and/orC9 gene.

The present invention also provides methods for preventing at least onesymptom, e.g., hemolysis, in a subject having a disorder that wouldbenefit from inhibiting or reducing the expression of a CFB, C3, and/orC9 gene, e.g., a complement component-associated disease, such asparoxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremicsyndrome (aHUS).

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofa CFB, C3, or C9 gene. The use of these iRNAs enables the targeteddegradation of mRNAs of the corresponding gene (CFB, C3, or C9 gene) inmammals. Very low dosages of the iRNAs of the invention, in particular,can specifically and efficiently mediate RNA interference (RNAi),resulting in significant inhibition of expression of the correspondinggene (CFB, C3, or C9 gene). Using cell-based assays, the presentinventors have demonstrated that iRNAs targeting these complementcomponent genes can mediate RNAi, resulting in significant inhibition ofexpression of a complement gene (i.e., CFB, C3, or C9). Thus, methodsand compositions including these iRNAs are useful for treating a subjecthaving a complement component-associated disease, such as paroxysmalnocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome(aHUS).

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of a complementgene (i.e., CFB, C3 or C9) as well as compositions, uses, and methodsfor treating subjects having diseases and disorders that would benefitfrom inhibition and/or reduction of the expression of these genes.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, the term “Complement Factor B,” used interchangeablywith the term “CFB,” refers to the well-known gene and polypeptide, alsoknown in the art as AHUS, BF, CFAB, BFD, FB, GBG, FBI12, B-Factor,Properdin, H2-Bf, Glycine-Rich Beta Glycoprotein, C3 Proaccelerator,Properdin Factor 2B, C3 Proactivator, PBF2, Glycine-RichBeta-Glycoprotein, C3/C5 Convertase, EC 3.4.21, and EC 3.4.21.473. Theterm “CFB” includes human CFB, the amino acid and nucleotide sequence ofwhich may be found in, for example, GenBank Accession No. GI:189181756;mouse CFB, the amino acid and nucleotide sequence of which may be foundin, for example, GenBank Accession Nos. GI:218156288 and GI:218156290;rat CFB, the amino acid and nucleotide sequence of which may be foundin, for example, GenBank Accession No. GI:218156284; and chimpanzee CFB,the amino acid and nucleotide sequence of which may be found in, forexample, GenBank Accession No. GI:57114201. The term “CFB” also includesMacaca fascicularis CFB, the amino acid and nucleotide sequence of whichmay be found in, for example, GenBank Accession No. GI:544428919 and inthe entry for the gene, ENSMMUP00000000985(locus=scaffold3881:47830:53620), in the Macaca genome project web site(http://macaquce.genomics.org.en/page/species/index.jsp).Additionalexamples of CFB mRNA sequences are readily available using, e.g.,GenBank, UniProt, OMIM, and the Macaca genome project web site.

Exemplary CFB nucleotide sequences may also be found in SEQ ID NOs:1-5,27, and 30. SEQ ID NOs:12-16, 33, and 36 are the antisense sequences ofSEQ ID NOs: 1-5, 27, and 30, respectively.

The term“CFB,” as used herein, also refers to naturally occurring DNAsequence variations of the CFB gene. Non-limiting examples of sequencevariations within the CFB gene include 1598A>G in exon 12, which resultsin a lysine being changed to an arginine at amino acid residue 533;858C>G in exon 6, which results in a phenylalanine being changed to aleucine at amino acid residue 286; and 967A>G in exon 7, which resultsin a lysine being changed to an alanine at amino acid residue 323(Tawadrous H. et al. (2010) Pediatr Nephrol. 25:947; Goicoechea de JorgeE et al. (2007) Proc Natl Acad Sci. USA 104:240). The term“CFB,” as usedherein, also refers to single nucleotide polymorphisms in the CFB gene.Numerous sequence variations within the CFB gene have been identifiedand may be found at, for example, NCBI dbSNP and UniProt (see, e.g.,ncbi.nlm.nih.gov/snp).

As used herein, the term “Complement Component 3,” used interchangeablywith the term “C3,” refers to the well-known gene and polypeptide, alsoknown in the art as ARMD9, C3a Anaphylatoxin, ASP, Complement ComponentC3a, C3a, Complement Component C3b, C3b, prepro-C3,Acylation-Stimulating Protein Cleavage Product, CPAMD1, Complement C3,C3 And PZP-Like Alpha-2-Macroglobulin Domain-Containing Protein 1,Complement Component C3, and AHUSS. The term “C3” includes human C3, theamino acid and nucleotide sequence of which may be found in, forexample, GenBank Accession No. GI:115298677; mouse C3, the amino acidand nucleotide sequence of which may be found in, for example, GenBankAccession No. GI:126518316; and rat C3, the amino acid and nucleotidesequence of which may be found in, for example, GenBank Accession No.GI:158138560. The term “C3” also includes Macaca fascicularis CFB, theamino acid and nucleotide sequence of which may be found in, forexample, GenBank Accession No. GI:544508182 and in the entry for thegene, ENSP00000245907 (locus=chr19:6921416:6963034), in the Macacagenome project web site(http://macaque.genomics.org.en/page/species/index.jsp). Additionalexamples of C3 mRNA sequences are readily available using, e.g.,GenBank, UniProt, OMIM, and the Macaca genome project web site.

Exemplary C3 nucleotide sequences may also be found in SEQ ID NOs:6-8,28, and 31. SEQ ID NOs:17-19, 34, and 37 are the antisense sequences ofSEQ ID NOs: 6-8, 28, and 31, respectively.

The term“C3,” as used herein, also refers to naturally occurring DNAsequence variations of the C3 gene. Numerous sequence variations withinthe C3 gene have been identified and may be found at, for example, NCBIdbSNP and UniProt (see, e.g., ncbi.nlm.nih.gov/snp).

As used herein, the term “Complement Component 9,” used interchangeablywith the term “C9,” refers to the well-known gene and polypeptide. Theterm “C9” includes human C9, the amino acid and nucleotide sequence ofwhich may be found in, for example, GenBank Accession No. GI:187608340;mouse C9, the amino acid and nucleotide sequence of which may be foundin, for example, GenBank Accession No. GI:15375311; and rat C9, theamino acid and nucleotide sequence of which may be found in, forexample, GenBank Accession No. GI:16924005. The term “C9” also includesMacaca fascicularis CFB, the amino acid and nucleotide sequence of whichmay be found in, for example, GenBank Accession No. GI:544436867 and inthe entry for the gene, isotig05361 (isogroup03350 length=2955numContigs=1), in the Macaca genome project web site(http://macaque.genomics.org.en/page/species/index.jsp). Additionalexamples of C3 mRNA sequences are readily available using, e.g.,GenBank, UniProt, OMIM, and the Macaca genome project web site.

Exemplary C9 nucleotide sequences may also be found in SEQ ID NOs:9-11,29, and 32. SEQ ID NOs:20-22, 35, and 38 are the antisense sequences ofSEQ ID NOs: 9-11, 29, and 32, respectively.

The term“C9,” as used herein, also refers to naturally occurring DNAsequence variations of the C9 gene. Numerous sequence variations withinthe C9 gene have been identified and may be found at, for example, NCBIdbSNP and UniProt (see, e.g., ncbi.nlm.nih.gov/snp).

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a CFB, C3, or C9 gene, including mRNA that is a product of RNAprocessing of a primary transcription product. In one embodiment, thetarget portion of the sequence will be at least long enough to serve asa substrate for iRNA-directed cleavage at or near that portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a CFB, C3, or C9 gene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of the target gene in a cell, e.g., a cell within asubject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., a CFB, C3,or C9 target mRNA sequence, to direct the cleavage of the target RNA.Without wishing to be bound by theory it is believed that long doublestranded RNA introduced into cells is broken down into siRNA by a TypeIII endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., a CFB, C3, or C9 gene. Accordingly,the term “siRNA” is also used herein to refer to an RNAi as describedabove.

In another embodiment, the RNAi agent may be a single-stranded siRNAthat is introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded siRNAs are described in U.S. Pat. No.8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double-stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., a CFB, C3, or C9 gene. In someembodiments of the invention, a double-stranded RNA (dsRNA) triggers thedegradation of a target RNA, e.g., an mRNA, through apost-transcriptional gene-silencing mechanism referred to herein as RNAinterference or RNAi. In general, the majority of nucleotides of eachstrand of a dsRNA molecule are ribonucleotides, but as described indetail herein, each or both strands can also include one or morenon-ribonucleotides, e.g., a deoxyribonucleotide and/or a modifiednucleotide. In addition, as used in this specification, an “RNAi agent”may include ribonucleotides with chemical modifications; an RNAi agentmay include substantial modifications at multiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, and/or a modified nucleobase. Thus, the termmodified nucleotide encompasses substitutions, additions or removal of,e.g., a functional group or atom, to internucleoside linkages, sugarmoieties, or nucleobases. The modifications suitable for use in theagents of the invention include all types of modifications disclosedherein or known in the art. Any such modifications, as used in a siRNAtype molecule, are encompassed by “RNAi agent” for the purposes of thisspecification and claims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised of separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded RNAi agent, i.e., no nucleotideoverhang. A “blunt ended” RNAi agent is a dsRNA that is double-strandedover its entire length, i.e., no nucleotide overhang at either end ofthe molecule. The RNAi agents of the invention include RNAi agents withnucleotide overhangs at one end (i.e., agents with one overhang and oneblunt end) or with nucleotide overhangs at both ends.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a CFB, C3, or C9 mRNA. As usedherein, the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., a CFB, C3, or C9 nucleotide sequence,as defined herein. Where the region of complementarity is not fullycomplementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding CFB, C3, or C9). For example, apolynucleotide is complementary to at least a part of a CFB mRNA if thesequence is substantially complementary to a non-interrupted portion ofan mRNA encoding CFB.

In general, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA”may include ribonucleotides with chemical modifications. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an iRNA molecule,are encompassed by “iRNA” for the purposes of this specification andclaims.

In one aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisense RNAmolecule that inhibits a target mRNA via an antisense inhibitionmechanism. The single-stranded antisense RNA molecule is complementaryto a sequence within the target mRNA. The single-stranded antisenseoligonucleotides can inhibit translation in a stoichiometric manner bybase pairing to the mRNA and physically obstructing the translationmachinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Thesingle-stranded antisense RNA molecule may be about 15 to about 30nucleotides in length and have a sequence that is complementary to atarget sequence. For example, the single-stranded antisense RNA moleculemay comprise a sequence that is at least about 15, 16, 17, 18, 19, 20,or more contiguous nucleotides from any one of the antisense sequencesdescribed herein.

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the iRNA or contacting a cell in vivo with the iRNA. The contactingmay be done directly or indirectly. Thus, for example, the RNAi agentmay be put into physical contact with the cell by the individualperforming the method, or alternatively, the RNAi agent may be put intoa situation that will permit or cause it to subsequently come intocontact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an RNAi agent and subsequently transplanted into a subject.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in CFB, C3, and/or C9 expression; a human at riskfor a disease, disorder or condition that would benefit from reductionin CFB, C3, and/or C9 expression; a human having a disease, disorder orcondition that would benefit from reduction in CFB, C3, and/or C9expression; and/or human being treated for a disease, disorder orcondition that would benefit from reduction in CFB, C3, and/or C9expression as described herein.

As used herein, the term “complement component-associated disease” is adisease or disorder that is caused by, or associated with complementactivation. The term “complement component-associated disease” includesa disease, disorder or condition that would benefit from reduction inCFB (i.e., a “CFB-associated disease”), C3 (i.e., a “C3-associateddisease”), and/or C9 (i.e., a “C9-associated disease”) expression. Suchdiseases are typically associated with inflammation and/or immune systemactivation, e.g., membrane attack complex-mediated lysis, anaphylaxis,and/or hemolysis. Non-limiting examples of complementcomponent-associated diseases include paroxysmal nocturnalhemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma,rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupusnephritis; ischemia-reperfusion injury; typical or infectious hemolyticuremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitisoptica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis(MS); macular degeneration (e.g., age-related macular degeneration(AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP)syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetalloss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetalloss; pre-eclampsia, traumatic brain injury, myasthenia gravis, coldagglutinin disease, dermatomyositis bullous pemphigoid, Shiga toxin E.coli-related hemolytic uremic syndrome, C3 neuropathy, anti-neutrophilcytoplasmic antibody-associated vasculitis (e.g., granulomatosis withpolyangiitis (previously known as Wegener granulomatosis), Churg-Strausssyndrome, and microscopic polyangiitis), humoral and vascular transplantrejection, graft dysfunction, myocardial infarction (e.g., tissue damageand ischemia in myocardial infarction), an allogenic transplant, sepsis(e.g., poor outcome in sepsis), Coronary artery disease,dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease,systemic inflammatory response sepsis, septic shock, spinal cord injury,glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis,pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasturesyndrome, Degos disease, antiphospholipid syndrome (APS), catastrophicAPS (CAPS), a cardiovascular disorder, myocarditis, a cerebrovasculardisorder, a peripheral (e.g., musculoskeletal) vascular disorder, arenovascular disorder, a mesenteric/enteric vascular disorder,vasculitis, Henoch-Schönlein purpura nephritis, systemic lupuserythematosus-associated vasculitis, vasculitis associated withrheumatoid arthritis, immune complex vasculitis, Takayasu's disease,dilated cardiomyopathy, diabetic angiopathy, Kawasaki's disease(arteritis), venous gas embolus (VGE), and restenosis following stentplacement, rotational atherectomy, and percutaneous transluminalcoronary angioplasty (PTCA) (see, e.g., Holers (2008) ImmunologicalReviews 223:300-316; Holers and Thurman (2004) Molecular Immunology41:147-152; U.S. Patent Publication No. 20070172483).

In one embodiment, a complement component-associated disease isparoxysmal nocturnal hemoglobinuria (PNH). The PNH may be classical PNHor PNH in the setting of another bone marrow failure syndrome and/ormyelodysplastic syndromes (MDS), e.g., cytopenias. In anotherembodiment, a complement component-associated disease is atypicalhemolytic uremic syndrome (aHUS). In yet another embodiment, acomplement component-associated disease is rheumatoid arthritis.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving a complement component-associated disease, is sufficient toeffect treatment of the disease (e.g., by diminishing, ameliorating ormaintaining the existing disease or one or more symptoms of disease).The “therapeutically effective amount” may vary depending on the RNAiagent or antibody, or antigen-binding fragment thereof, how the agent isadministered, the disease and its severity and the history, age, weight,family history, genetic makeup, the types of preceding or concomitanttreatments, if any, and other individual characteristics of the subjectto be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA agent that, when administered to a subjecthaving a complement component-associate disease but not yet (orcurrently) experiencing or displaying symptoms of the disease, and/or asubject at risk of developing a complement component-associated disease,e.g., a subject having a graft and/or transplant, e.g., a sensitized orallogenic recipient, a subject having sepsis, and/or a subject having amyocardial infarction, is sufficient to prevent or ameliorate thedisease or one or more symptoms of the disease. Ameliorating the diseaseincludes slowing the course of the disease or reducing the severity oflater-developing disease. The “prophylactically effective amount” mayvary depending on the iRNA agent, how the agent is administered, thedegree of risk of disease, and the history, age, weight, family history,genetic makeup, the types of preceding or concomitant treatments, ifany, and other individual characteristics of the patient to be treated.

A “therapeutically effective amount” or “prophylactically effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. iRNA agents employed in the methods of thepresent invention may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, urine, lymph,cerebrospinal fluid, ocular fluids, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissue(or subcomponents thereof) derived from the subject.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of acomplement component gene. In one embodiment, the iRNA agent includesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of a CFB gene in a cell, such as a cell within a subject,e.g., a mammal, such as a human having a complement component-associateddisease as described herein, e.g., PNH. In another embodiment, the iRNAagent includes double-stranded ribonucleic acid (dsRNA) molecules forinhibiting the expression of a C3 gene in a cell, such as a cell withina subject, e.g., a mammal, such as a human having a complementcomponent-associated disease as described herein, e.g., PNH. In afurther embodiment, the iRNA agent includes double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of a C9 gene in acell, such as a cell within a subject, e.g., a mammal, such as a humanhaving a complement component-associated disease as described herein,e.g., PNH. The dsRNA includes an antisense strand having a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of a target gene, i.e., CFB, C3, or C9 gene.The region of complementarity is about 30 nucleotides or less in length(e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18nucleotides or less in length). Upon contact with a cell expressing thetarget gene, the iRNA inhibits the expression of the target gene (e.g.,a human, a primate, a non-primate, or a bird CFB, C3, or C9 gene) by atleast about 10% as assayed by, for example, a PCR or branched DNA(bDNA)-based method, or by a protein-based method, such as byimmunofluorescence analysis, using, for example, Western Blotting orflowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of a CFB, C3,or C9 gene. The other strand (the sense strand) includes a region thatis complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

In some embodiments, the dsRNA is between about 15 and about 20nucleotides in length, or between about 25 and about 30 nucleotides inlength. In one embodiment, an RNAi agent of the invention is a dsRNA of24-30 nucleotides that interacts with a target RNA sequence, i.e., aCFB, C3, or C9 target mRNA sequence, to direct the cleavage of thetarget RNA. In general, the dsRNA is long enough to serve as a substratefor the Dicer enzyme. For example, it is well-known in the art thatdsRNAs longer than about 21-23 nucleotides in length may serve assubstrates for Dicer. As the ordinarily skilled person will alsorecognize, the region of an RNA targeted for cleavage will most often bepart of a larger RNA molecule, often an mRNA molecule. Where relevant, a“part” of an mRNA target is a contiguous sequence of an mRNA target ofsufficient length to allow it to be a substrate for RNAi-directedcleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target CFB, C3, or C9 expression is not generated in the target cellby cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double-stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence.

In one embodiment, a dsRNA of the invention targeting CFB includes asense strand selected from the group of sequences provided in any one ofTables 3 and 4, and the corresponding antisense strand of the sensestrand is selected from the group of sequences of any one of Tables 3and 4. In this aspect, one of the two sequences is complementary to theother of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of a CFB gene. As such, in this aspect, a dsRNA will includetwo oligonucleotides, where one oligonucleotide is described as thesense strand in any one of Tables 3 and 4, and the secondoligonucleotide is described as the corresponding antisense strand ofthe sense strand in any one of Tables 3 and 4. In one embodiment, thesubstantially complementary sequences of the dsRNA are contained onseparate oligonucleotides. In another embodiment, the substantiallycomplementary sequences of the dsRNA are contained on a singleoligonucleotide.

In one embodiment, a dsRNA of the invention targeting C3 includes asense strand selected from the group of sequences provided in any one ofTables 5 and 6, and the corresponding antisense strand of the sensestrand is selected from the group of sequences of any one of Tables 5and 6. In this aspect, one of the two sequences is complementary to theother of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of a C3 gene. As such, in this aspect, a dsRNA will includetwo oligonucleotides, where one oligonucleotide is described as thesense strand in any one of Tables 5 and 6, and the secondoligonucleotide is described as the corresponding antisense strand ofthe sense strand in any one of Tables 5 and 6. In one embodiment, thesubstantially complementary sequences of the dsRNA are contained onseparate oligonucleotides. In another embodiment, the substantiallycomplementary sequences of the dsRNA are contained on a singleoligonucleotide.

In one embodiment, a dsRNA of the invention targeting C9 includes asense strand selected from the group of sequences provided in any one ofTables 7 and 8, and the corresponding antisense strand of the sensestrand is selected from the group of sequences of any one of Tables 7and 8. In this aspect, one of the two sequences is complementary to theother of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of a C9 gene. As such, in this aspect, a dsRNA will includetwo oligonucleotides, where one oligonucleotide is described as thesense strand in any one of Tables 7 and 8, and the secondoligonucleotide is described as the corresponding antisense strand ofthe sense strand in any one of Tables 7 and 8. In one embodiment, thesubstantially complementary sequences of the dsRNA are contained onseparate oligonucleotides. In another embodiment, the substantiallycomplementary sequences of the dsRNA are contained on a singleoligonucleotide.

It will be understood that, although some of the sequences in Tables 3-8are described as modified and/or conjugated sequences, the RNA of theiRNA of the invention e.g., a dsRNA of the invention, may comprise anyone of the sequences set forth in Tables 3-8 that is un-modified,un-conjugated, and/or modified and/or conjugated differently thandescribed therein.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., EMBO 2001, 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 3-8 dsRNAsdescribed herein can include at least one strand of a length ofminimally 21 nucleotides. It can be reasonably expected that shorterduplexes having one of the sequences of any one of Tables 3-8 minus onlya few nucleotides on one or both ends can be similarly effective ascompared to the dsRNAs described above. Hence, dsRNAs having a sequenceof at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotidesderived from one of the sequences of any one of Tables 3-8, anddiffering in their ability to inhibit the expression of the target geneby not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated to be within the scope ofthe present invention.

In addition, the RNAs provided in any one of Tables 3 and 4 identify asite(s) in a CFB transcript that is susceptible to RISC-mediatedcleavage. Similarly, the RNAs provided in any one of Tables 5 and 6identify a site(s) in a C3 transcript that is susceptible toRISC-mediated cleavage, and the RNAs provided in any one of Tables 7 and8 identify a site(s) in a C9 transcript that is susceptible toRISC-mediated cleavage. As such, the present invention further featuresiRNAs that target within one of these sites. As used herein, an iRNA issaid to target within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least about 15 contiguousnucleotides from one of the sequences provided in any one of Tables 3-8coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in the target gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in any one of Tables 3-8represent effective target sequences, it is contemplated that furtheroptimization of inhibition efficiency can be achieved by progressively“walking the window” one nucleotide upstream or downstream of the givensequences to identify sequences with equal or better inhibitioncharacteristics.

Further, it is contemplated that for any sequence identified, e.g., inany one of Tables 3-8, further optimization could be achieved bysystematically either adding or removing nucleotides to generate longeror shorter sequences and testing those sequences generated by walking awindow of the longer or shorter size up or down the target RNA from thatpoint. Again, coupling this approach to generating new candidate targetswith testing for effectiveness of iRNAs based on those target sequencesin an inhibition assay as known in the art and/or as described hereincan lead to further improvements in the efficiency of inhibition.Further still, such optimized sequences can be adjusted by, e.g., theintroduction of modified nucleotides as described herein or as known inthe art, addition or changes in overhang, or other modifications asknown in the art and/or discussed herein to further optimize themolecule (e.g., increasing serum stability or circulating half-life,increasing thermal stability, enhancing transmembrane delivery,targeting to a particular location or cell type, increasing interactionwith silencing pathway enzymes, increasing release from endosomes) as anexpression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of, e.g., a CFB gene, generally doesnot contain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of a target gene, e.g., a CFB, C3, or C9gene. Consideration of the efficacy of iRNAs with mismatches ininhibiting expression of a target gene is important, especially if theparticular region of complementarity in a target gene is known to havepolymorphic sequence variation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is un-modified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA ofthe invention are modified iRNAs of the invention in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH², and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chico. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT). 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclicsugar moities. A “bicyclic sugar” is a furanosyl ring modified by thebridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleosidehaving a sugar moiety comprising a bridge connecting two carbon atoms ofthe sugar ring, thereby forming a bicyclic ring system. In certainembodiments, the bridge connects the 4′-carbon and the 2′-carbon of thesugar ring. Thus, in some embodiments an agent of the invention mayinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. In otherwords, an LNA is a nucleotide comprising a bicyclic sugar moietycomprising a 4′-CH₂—O-2′ bridge. This structure effectively “locks” theribose in the 3′-endo structural conformation. The addition of lockednucleic acids to siRNAs has been shown to increase siRNA stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in thepolynucleotides of the invention include without limitation nucleosidescomprising a bridge between the 4′ and the 2′ ribosyl ring atoms. Incertain embodiments, the antisense polynucleotide agents of theinvention include one or more bicyclic nucleosides comprising a 4′ to 2′bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, includebut are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′;4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′(see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative U.S. Patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may alsoinclude a hydroxymethyl substituted nucleotide. A “hydroxymethylsubstituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, alsoreferred to as an “unlocked nucleic acid” (“UNA”) modification

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference. Potentially stabilizing modifications to the ends of RNAmolecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol(Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6),N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine(ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double-stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in U.S. Provisional Application No. 61/561,710, filed onNov. 18, 2011, or in PCT/US2012/065691, the entire contents of each ofwhich are incorporated herein by reference.

As shown herein and in Provisional Application No. 61/561,710 or inPCT/US2012/065691, a superior result may be obtained by introducing oneor more motifs of three identical modifications on three consecutivenucleotides into a sense strand and/or antisense strand of an RNAiagent, particularly at or near the cleavage site. In some embodiments,the sense strand and antisense strand of the RNAi agent may otherwise becompletely modified. The introduction of these motifs interrupts themodification pattern, if present, of the sense and/or antisense strand.The RNAi agent may be optionally conjugated with a GalNAc derivativeligand, for instance on the sense strand. The resulting RNAi agentspresent superior gene silencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded RNAi agent arecompletely modified to have one or more motifs of three identicalmodifications on three consecutive nucleotides at or near the cleavagesite of at least one strand of an RNAi agent, the gene silencingactivity of the RNAi agent was superiorly enhanced.

Accordingly, the invention provides double-stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., a CFB, C3, or C9gene) in vivo. The RNAi agent comprises a sense strand and an antisensestrand. Each strand of the RNAi agent may range from 12-30 nucleotidesin length. For example, each strand may be between 14-30 nucleotides inlength, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides inlength, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides inlength, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TT can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′ end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′ end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand. When the 2 nucleotide overhang is at the3′-end of the antisense strand, there may be two phosphorothioateinternucleotide linkages between the terminal three nucleotides, whereintwo of the three nucleotides are the overhang nucleotides, and the thirdnucleotide is a paired nucleotide next to the overhang nucleotide. Inone embodiment, the RNAi agent additionally has two phosphorothioateinternucleotide linkages between the terminal three nucleotides at boththe 5′-end of the sense strand and at the 5′-end of the antisensestrand. In one embodiment, every nucleotide in the sense strand and theantisense strand of the RNAi agent, including the nucleotides that arepart of the motifs are modified nucleotides. In one embodiment eachresidue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g.,in an alternating motif. Optionally, the RNAi agent further comprises aligand (preferably GalNAC₃).

In one embodiment, the RNAi agent comprises a sense and an antisensestrand, wherein the sense strand is 25-30 nucleotide residues in length,wherein starting from the 5′ terminal nucleotide (position 1) positions1 to 23 of the first strand comprise at least 8 ribonucleotides; theantisense strand is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of sense strand to form aduplex; wherein at least the 3 ‘ terminal nucleotide of antisense strandis unpaired with sense strand, and up to 6 consecutive 3’ terminalnucleotides are unpaired with sense strand, thereby forming a 3′ singlestranded overhang of 1-6 nucleotides; wherein the 5′ terminus ofantisense strand comprises from 10-30 consecutive nucleotides which areunpaired with sense strand, thereby forming a 10-30 nucleotide singlestranded 5′ overhang; wherein at least the sense strand 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of antisensestrand when sense and antisense strands are aligned for maximumcomplementarity, thereby forming a substantially duplexed region betweensense and antisense strands; and antisense strand is sufficientlycomplementary to a target RNA along at least 19 ribonucleotides ofantisense strand length to reduce target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell; and whereinthe sense strand contains at least one motif of three 2′-F modificationson three consecutive nucleotides, where at least one of the motifsoccurs at or near the cleavage site. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region region which is at least 25 nucleotides in length, andthe second strand is sufficiently complementary to a target mRNA alongat least 19 nucleotide of the second strand length to reduce target geneexpression when the RNAi agent is introduced into a mammalian cell, andwherein dicer cleavage of the RNAi agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the RNAi agentfurther comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For an RNAi agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradjacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking 0 of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking 0position may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisense strand may start with “BBAABBAA” from5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages. In one embodiment, the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′-end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT). In another embodiment, the nucleotide at the3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment,there is a short sequence of deoxy-thymine nucleotides, for example, twodT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented byformula (I):

5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of - the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

5′ n_(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n_(q) 3′  (Ib);

5′ n_(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n_(q) 3′  (Ic); or

5′ n_(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n_(q) 3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

5′ n_(p)-N_(a)—YYY—N_(a)-n_(q) 3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

5′n_(q′)-N_(a)′—(Z′Z′Z′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(X′X′X′)_(l)—N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

5′ n_(q)′-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(a)′-n_(p)′ 3′  (IIb);

5′ n_(q)′-N_(a)′—Y′Y′Y′—N_(b)′—X′X′X′-n_(p)′ 3′  (IIc); or

5′ n_(q)′-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(b)′—X′X′X′—N_(a)′-n_(p′)3′  (IId).

When the antisense strand is represented by formula (lib), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may berepresented by the formula:

5′ n_(p)′-N_(a)′—Y′Y′Y′—N_(a)-n_(q′) 3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. Forexample, each nucleotide of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′,Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′ n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q) 3′

antisense: 3′n_(p)′-N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′  (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or maynot be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

5′ n_(p)-N_(a)—YYY—N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′—Y′Y′Y′—N_(a)′-n_(q)′ 5′   (IIIa)

5′ n_(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)′n_(q)′ 5′   (IIIb)

5′ n_(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n_(q)′ 5′   (IIIc)

5′ n_(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)-n_(q)′ 5′  (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b) independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In another embodiment, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the RNAi agent that containsconjugations of one or more carbohydrate moieties to a RNAi agent canoptimize one or more properties of the RNAi agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of the RNAiagent. For example, the ribose sugar of one or more ribonucleotidesubunits of a dsRNA agent can be replaced with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier to which is attached acarbohydrate ligand. A ribonucleotide subunit in which the ribose sugarof the subunit has been so replaced is referred to herein as a ribosereplacement modification subunit (RRMS). A cyclic carrier may be acarbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methodsof the invention is an agent selected from the group of agents listed inany one of Tables 3-8. These agents may further comprise a ligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 23). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 24) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 25) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 26)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of;

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other isa hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.

These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different; P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A),P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A),T^(5B), T^(5C) are each independently for each occurrence absent, CO,NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherin one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923).

Representative United States patents that teach the preparation of suchRNA conjugates have been listed above. Typical conjugation protocolsinvolve the synthesis of an RNAs bearing an aminolinker at one or morepositions of the sequence. The amino group is then reacted with themolecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction can be performed either with the RNAstill bound to the solid support or following cleavage of the RNA, insolution phase. Purification of the RNA conjugate by HPLC typicallyaffords the pure conjugate.

V. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a complement component-associated disease asdescribed herein) can be achieved in a number of different ways. Forexample, delivery may be performed by contacting a cell with an iRNA ofthe invention either in vitro or in vivo. In vivo delivery may also beperformed directly by administering a composition comprising an iRNA,e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may beperformed indirectly by administering one or more vectors that encodeand direct the expression of the iRNA. These alternatives are discussedfurther below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting a CFB, C3, or C9 gene can be expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can betransient (on the order of hours to weeks) or sustained (weeks to monthsor longer), depending upon the specific construct used and the targettissue or cell type. These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can be anintegrating or non-integrating vector. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment.

Delivery of iRNA expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from the patient followed by reintroduction into thepatient, or by any other means that allows for introduction into adesired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inventionis a pox virus such as a vaccinia virus, for example an attenuatedvaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such asfowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of a CFB, C3, and/or C9 gene, e.g. a complementcomponent-associated disease as descried herein. Such pharmaceuticalcompositions are formulated based on the mode of delivery. One exampleis compositions that are formulated for systemic administration viaparenteral delivery, e.g., by subcutaneous (SC) or intravenous (IV)delivery. Another example is compositions that are formulated for directdelivery into the brain parenchyma, e.g., by infusion into the brain,such as by continuous pump infusion. The pharmaceutical compositions ofthe invention may be administered in dosages sufficient to inhibitexpression of the target gene. In general, a suitable dose of an iRNA ofthe invention will be in the range of about 0.001 to about 200.0milligrams per kilogram body weight of the recipient per day, generallyin the range of about 1 to 50 mg per kilogram body weight per day. Forexample, the dsRNA can be administered at about 0.01 mg/kg, about 0.05mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg,about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40mg/kg, or about 50 mg/kg per single dose.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In another embodiment, the dsRNA is administered at a dose of about 0.1to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. In one embodiment, the dsRNA isadministered at a dose of about 10 mg/kg to about 30 mg/kg. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered, e.g., subcutaneously orintravenously, a single therapeutic amount of iRNA, such as about 0.1,0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375,0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65,0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925,0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22,22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29,29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In some embodiments, subjects are administered, e.g., subcutaneously orintravenously, multiple doses of a therapeutic amount of iRNA, such as adose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325,0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6,0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875,0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28,28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. A multi-dose regimine mayinclude administration of a therapeutic amount of iRNA daily, such asfor two days, three days, four days, five days, six days, seven days, orlonger.

In other embodiments, subjects are administered, e.g., subcutaneously orintravenously, a repeat dose of a therapeutic amount of iRNA, such as adose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325,0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6,0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875,0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28,28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. A repeat-dose regiminemay include administration of a therapeutic amount of iRNA on a regularbasis, such as every other day, every third day, every fourth day, twicea week, once a week, every other week, or once a month.

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as a disorder that wouldbenefit from reduction in the expression of CFB, C3, or C9. Such modelscan be used for in vivo testing of iRNA, as well as for determining atherapeutically effective dose. Suitable mouse models are known in theart and include, for example, collagen-induced arthritis mouse model(Courtenay, J. S., et al. (1980) Nature 283, 666-668), myocardialischemia (Homeister J W and Lucchesi B R (1994) Annu Rev PharmacolToxicol 34:17-40), ovalbumin induced asthma mouse models (e.g.,Tomkinson A., et al. (2001). J. Immunol. 166, 5792-5800), (NZB×NZW)F1,MRL/Fas^(lpr) (MRL/lpr) and BXSB mouse models (Theofilopoulos, A. N. andKono, D. H. 1999. Murine lupus models: gene-specific and genome-widestudies. In Lahita R. G., ed., Systemic Lupus Erythematosus, 3rd edn, p.145. Academic Press, San Diego, Calif.), mouse aHUS model (Goicoechea deJorge et al. (2011) The development of atypical hemolytic uremicsyndrome depends on complement C5, J Am Soc Nephrol 22:137-145.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging RNAi agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated RNAi agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of RNAi agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer RNAi agent into the skin. In some implementations,liposomes are used for delivering RNAi agent to epidermal cells and alsoto enhance the penetration of RNAi agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith RNAi agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include RNAi agentcan be delivered, for example, subcutaneously by infection in order todeliver RNAi agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40%2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98-4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/ PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-1,2-Dilinolenyloxy- DLinDMA/DPPC/ 1 N,N-dimethylaminopropaneCholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA ~7:12-XTC 2,2-Dilinoleyl-4- XTC/DPPC/ dimethylaminoethyl-[1,3]-Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1LNP05 2,2-Dilinoleyl-4- XTC/DSPC/ dimethylaminoethyl-[1,3]-Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1LNP06 2,2-Dilinoleyl-4- XTC/DSPC/ dimethylaminoethyl-[1,3]-Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1LNP07 2,2-Dilinoleyl-4- XTC/DSPC/ dimethylaminoethyl-[1,3]-Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~6:1LNP08 2,2-Dilinoleyl-4- XTC/DSPC/ dimethylaminoethyl-[1,3]-Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~11:1LNP09 2,2-Dilinoleyl-4- XTC/DSPC/ dimethylaminoethyl-[1,3]-Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1LNP10 (3aR,5s,6aS)-N,N- ALN100/DSPC/ dimethyl-2,2-di((9Z,12Z)-Cholesterol/PEG-DMG octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxo1- 5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)- MC-3/DSPC/ heptatriaconta-6,9,28,31-Cholesterol/PEG-DMG tetraen-19-yl 4- 50/10/38.5/1.5(dimethylamino)butanoate Lipid:siRNA 10:1 (MC3) LNP121,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/ hydroxydodecyl)amino)ethyl)(2-Cholesterol/PEG-DMG hydroxydodecyl)ami- 50/10/38.5/1.5no)ethyl)piperazin-1- Lipid:siRNA 10:1 yl)ethylazanediyedidodecan- 2-ol(Tech G1) LNP13 XTC XTC/DSPC/ Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA:33:1 LNP14 MC3 MC3/DSPC/ Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15MC3 MC3/DSPC/Chol/PEG- DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA:11:1 LNP16 MC3 MC3/DSPC/ Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1LNP17 MC3 MC3/DSPC/ Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18MC3 MC3/DSPC/ Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/ Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/ Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/ Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1DSPC: distearoylphosphatidylcholineDPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO₂Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO₂Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g). Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa₂SO₄ solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO₃solution (1×50 mL). The organic layer was then dried over anhyd. Na₂SO₄and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na₂SO₃ and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an.Na₂SO₄ and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: —6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]-266.3,[M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na₂SO₄ then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR δ=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include polyamino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and antioxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid.

Antioxidants are also commonly added to emulsion formulations to preventdeterioration of the formulation. Antioxidants used can be free radicalscavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

an RNAi agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™_50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-RNAi mechanism and which are useful intreating a hemolytic disorder. Examples of such agents include, but arenot limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent. In addition, other substancescommonly used to protect the liver, such as silymarin, can also be usedin conjunction with the iRNAs described herein. Other agents useful fortreating liver diseases include telbivudine, entecavir, and proteaseinhibitors such as telaprevir and other disclosed, for example, in Tunget al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116,and 2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby CFB, C3, and/or C9 expression. In any event, the administeringphysician can adjust the amount and timing of iRNA administration on thebasis of results observed using standard measures of efficacy known inthe art or described herein.

VII. Methods for Inhibiting Complement Component Expression

The present invention provides methods of inhibiting expression of acomplement component as described herein. In one aspect, the presentinvention provides methods of inhibiting expression of CFB in a cell.The methods include contacting a cell with an RNAi agent, e.g., a doublestranded RNAi agent, in an amount effective to inhibit expression of theCFB in the cell, thereby inhibiting expression of the CFB in the cell.

The present invention also provides methods of inhibiting expression ofC3 in a cell. The methods include contacting a cell with an RNAi agent,e.g., a double stranded RNAi agent, in an amount effective to inhibitexpression of the C3 in the cell, thereby inhibiting expression of theC3 in the cell.

In addition, the present invention provides methods of inhibitingexpression of C9 in a cell. The methods include contacting a cell withan RNAi agent, e.g., a double stranded RNAi agent, in an amounteffective to inhibit expression of the C9 in the cell, therebyinhibiting expression of the C9 in the cell.

Contacting of a cell with a double stranded RNAi agent may be done invitro or in vivo. Contacting a cell in vivo with the RNAi agent includescontacting a cell or group of cells within a subject, e.g., a humansubject, with the RNAi agent. Combinations of in vitro and in vivomethods of contacting are also possible. Contacting may be direct orindirect, as discussed above. Furthermore, contacting a cell may beaccomplished via a targeting ligand, including any ligand describedherein or known in the art. In preferred embodiments, the targetingligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, or any otherligand that directs the RNAi agent to a site of interest, e.g., theliver of a subject.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating” and other similar terms, andincludes any level of inhibition.

The phrase “inhibiting expression of a CFB” is intended to refer toinhibition of expression of any CFB gene (such as, e.g., a mouse CFBgene, a rat CFB gene, a monkey CFB gene, or a human CFB gene) as well asvariants or mutants of a CFB gene. Thus, the CFB gene may be a wild-typeCFB gene, a mutant CFB gene, or a transgenic CFB gene in the context ofa genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a CFB gene” includes any level of inhibitionof a CFB gene, e.g., at least partial suppression of the expression of aCFB gene. The expression of the CFB gene may be assessed based on thelevel, or the change in the level, of any variable associated with CFBgene expression, e.g., CFB mRNA level, CFB protein level, or, forexample, CH₅₀ activity as a measure of total hemolytic complement, AH₅₀to measure the hemolytic activity of the alternate pathway ofcomplement, and/or lactate dehydrogenase (LDH) levels as a measure ofintravascular hemolysis, and/or hemoglobin levels. Levels of C3, C9, C5,C5a, C5b, and soluble C5b-9 complex may also be measured to assess CFBexpression. Inhibition may be assessed by a decrease in an absolute orrelative level of one or more of these variables compared with a controllevel. The control level may be any type of control level that isutilized in the art, e.g., a pre-dose baseline level, or a leveldetermined from a similar subject, cell, or sample that is untreated ortreated with a control (such as, e.g., buffer only control or inactiveagent control).

The phrase “inhibiting expression of a C3” is intended to refer toinhibition of expression of any C3 gene (such as, e.g., a mouse C3 gene,a rat C3 gene, a monkey C3 gene, or a human C3 gene) as well as variantsor mutants of a C3 gene. Thus, the C3 gene may be a wild-type C3 gene, amutant C3 gene, or a transgenic C3 gene in the context of a geneticallymanipulated cell, group of cells, or organism.

“Inhibiting expression of a C3 gene” includes any level of inhibition ofa C3 gene, e.g., at least partial suppression of the expression of a C3gene. The expression of the C3 gene may be assessed based on the level,or the change in the level, of any variable associated with C3 geneexpression, e.g., C3 mRNA level, C3 protein level, or, for example, CH₅₀activity as a measure of total hemolytic complement, AH₅₀ to measure thehemolytic activity of the alternate pathway of complement, and/orlactate dehydrogenase (LDH) levels as a measure of intravascularhemolysis, and/or hemoglobin levels. Levels of CFB, C9, C5, C5a, C5b,and soluble C5b-9 complex may also be measured to assess C3 expression.Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more of these variables compared with a control level.The control level may be any type of control level that is utilized inthe art, e.g., a pre-dose baseline level, or a level determined from asimilar subject, cell, or sample that is untreated or treated with acontrol (such as, e.g., buffer only control or inactive agent control).

The phrase “inhibiting expression of a C9” is intended to refer toinhibition of expression of any C9 gene (such as, e.g., a mouse C9 gene,a rat C9 gene, a monkey C9 gene, or a human C9 gene) as well as variantsor mutants of a C9 gene. Thus, the C9 gene may be a wild-type C9 gene, amutant C9 gene, or a transgenic C9 gene in the context of a geneticallymanipulated cell, group of cells, or organism.

“Inhibiting expression of a C9 gene” includes any level of inhibition ofa C9 gene, e.g., at least partial suppression of the expression of a C9gene. The expression of the C9 gene may be assessed based on the level,or the change in the level, of any variable associated with C9 geneexpression, e.g., C9 mRNA level, C9 protein level, or, for example, CH₅₀activity as a measure of total hemolytic complement, AH₅₀ to measure thehemolytic activity of the alternate pathway of complement, and/orlactate dehydrogenase (LDH) levels as a measure of intravascularhemolysis, and/or hemoglobin levels. Levels of CFB, C3, C5, C5a, C5b,and soluble C5b-9 complex may also be measured to assess C9 expression.Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more of these variables compared with a control level.The control level may be any type of control level that is utilized inthe art, e.g., a pre-dose baseline level, or a level determined from asimilar subject, cell, or sample that is untreated or treated with acontrol (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of atarget gene, e.g., CFB, C3, or C9 gene, is inhibited by at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%.at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%.

Inhibition of the expression of the target gene, e.g., a CFB, C3, or C9,gene may be manifested by a reduction of the amount of mRNA expressed bya first cell or group of cells (such cells may be present, for example,in a sample derived from a subject) in which a target gene istranscribed and which has or have been treated (e.g., by contacting thecell or cells with an RNAi agent of the invention, or by administeringan RNAi agent of the invention to a subject in which the cells are orwere present) such that the expression of a target gene is inhibited, ascompared to a second cell or group of cells substantially identical tothe first cell or group of cells but which has not or have not been sotreated (control cell(s)). In preferred embodiments, the inhibition isassessed by expressing the level of mRNA in treated cells as apercentage of the level of mRNA in control cells, using the followingformula:

${\frac{( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} ) - ( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} )}{( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} )} \cdot 100}\%$

Inhibition of the expression of a complement component protein may bemanifested by a reduction in the level of the protein that is expressedby a cell or group of cells (e.g., the level of protein expressed in asample derived from a subject). As explained above for the assessment ofmRNA suppression, the inhibition of protein expression levels in atreated cell or group of cells may similarly be expressed as apercentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of a target gene includes a cell or groupof cells that has not yet been contacted with an RNAi agent of theinvention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

The level of CFB, C3, or C9 mRNA that is expressed by a cell or group ofcells may be determined using any method known in the art for assessingmRNA expression. In one embodiment, the level of expression of CFB, C3,and/or C9 in a sample is determined by detecting a transcribedpolynucleotide, or portion thereof, e.g., mRNA of the CFB, C3, and/or C9gene. RNA may be extracted from cells using RNA extraction techniquesincluding, for example, using acid phenol/guanidine isothiocyanateextraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen)or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizingribonucleic acid hybridization include nuclear run-on assays, RT-PCR,RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035),Northern blotting, in situ hybridization, and microarray analysis.

In one embodiment, the level of expression of CFB, C3, and/or C9 isdetermined using a nucleic acid probe. The term “probe”, as used herein,refers to any molecule that is capable of selectively binding to aspecific CFB, C3, or C9. Probes can be synthesized by one of skill inthe art, or derived from appropriate biological preparations. Probes maybe specifically designed to be labeled. Examples of molecules that canbe utilized as probes include, but are not limited to, RNA, DNA,proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize, e.g.,specifically hybridize, to CFB, C3, or C9 mRNA. In one embodiment, themRNA is immobilized on a solid surface and contacted with a probe, forexample by running the isolated mRNA on an agarose gel and transferringthe mRNA from the gel to a membrane, such as nitrocellulose. In analternative embodiment, the probe(s) are immobilized on a solid surfaceand the mRNA is contacted with the probe(s), for example, in anAffymetrix gene chip array. A skilled artisan can readily adapt knownmRNA detection methods for use in determining the level of CFB, C3,and/or C9 mRNA.

An alternative method for determining the level of expression of CFB,C3, and/or C9 in a sample involves the process of nucleic acidamplification and/or reverse transcriptase (to prepare cDNA) of forexample mRNA in the sample, e.g., by RT-PCR (the experimental embodimentset forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chainreaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), selfsustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh etal. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al. (1988) Bio/Technology 6:1197), rolling circlereplication (Lizardi et al., U.S. Pat. No. 5,854,033) or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. In particular aspects of the invention, the level of expressionof CFB, C3, and/or C9 is determined by quantitative fluorogenic RT-PCR(i.e., the TaqMan™ System).

The expression levels of CFB, C3, and/or C9 mRNA may be monitored usinga membrane blot (such as used in hybridization analysis such asNorthern, Southern, dot, and the like), or microwells, sample tubes,gels, beads or fibers (or any solid support comprising bound nucleicacids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195and 5,445,934, which are incorporated herein by reference. Thedetermination of PCSK9 expression level may also comprise using nucleicacid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of CFB, C3, and/or C9 protein expression may be determinedusing any method known in the art for the measurement of protein levels.Such methods include, for example, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, fluid or gelprecipitin reactions, absorption spectroscopy, a colorimetric assays,spectrophotometric assays, flow cytometry, immunodiffusion (single ordouble), immunoelectrophoresis, Western blotting, radioimmunoassay(RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescentassays, electrochemiluminescence assays, and the like.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, lymph, urine,cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissuederived from the subject.

In some embodiments of the methods of the invention, the RNAi agent isadministered to a subject such that the RNAi agent is delivered to aspecific site within the subject. The inhibition of expression of CFB,C3, and/or C9 may be assessed using measurements of the level or changein the level of CFB, C3, and/or C9 mRNA and/or CFB, C3, and/or C9protein in a sample derived from fluid or tissue from the specific sitewithin the subject. In preferred embodiments, the site is the liver. Thesite may also be a subsection or subgroup of cells from any one of theaforementioned sites. The site may also include cells that express aparticular type of receptor.

VIII. Methods for Treating or Preventing a ComplementComponent-Associated Disease

The present invention provides therapeutic and prophylactic methodswhich include administering to a subject having a complementcomponent-associated disease, as described herein, e.g., PNH or aHUS, aniRNA agent, pharmaceutical compositions comprising an iRNA agent, orvector comprising an iRNA of the invention.

It is to be understood, that any of the methods of the invention may bepracticed with a single iRNA agent of the invention or a combination ofiRNA agents of the invention. For example, in some aspects, the methods(and uses) of the invention include using an iRNA agent targeting a CFBgene and an iRNA agent targeting a C3 gene. In some aspects, the methods(and uses) of the invention include using an iRNA agent targeting a CFBgene and an iRNA agent targeting a C9 gene. In some aspects, the methods(and uses) of the invention include using an iRNA agent targeting a C3gene and an iRNA agent targeting a C9 gene. In other aspects, themethods (and uses) of the invention include using an iRNA agenttargeting a CFB gene, an iRNA agent targeting a C3 gene, and an iRNAagent targeting a C9 gene. In some aspects of the invention, the methodswhich include either a single iRNA agent of the invention or acombination of iRNA agents, further include administering to the subjectone or more additional therapeutic agents such as, for example, Soliris®(as further described below).

In one aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in CFBexpression, e.g., “a complement component-associated disease,” e.g.,PNH, aHUS, or rheumatoid arthritis. The treatment methods (and uses) ofthe invention include administering to the subject, e.g., a human, atherapeutically effective amount of an iRNA agent targeting a CFB geneor a pharmaceutical composition comprising an iRNA agent targeting a CFBgene, thereby treating the subject having a disorder that would benefitfrom reduction in CFB expression.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in C3expression, e.g., “a complement component-associated disease,” e.g.,PNH, aHUS, or rheumatoid arthritis. The treatment methods (and uses) ofthe invention include administering to the subject, e.g., a human, atherapeutically effective amount of an iRNA agent targeting a C3 gene ora pharmaceutical composition comprising an iRNA agent targeting a C3gene, thereby treating the subject having a disorder that would benefitfrom reduction in C3 expression.

In a further aspect, the present invention provides methods of treatinga subject having a disorder that would benefit from reduction in C9expression, e.g., “a complement component-associated disease,” e.g.,PNH, aHUS, or rheumatoid arthritis. The treatment methods (and uses) ofthe invention include administering to the subject, e.g., a human, atherapeutically effective amount of an iRNA agent targeting a C9 gene ora pharmaceutical composition comprising an iRNA agent targeting a C9gene, thereby treating the subject having a disorder that would benefitfrom reduction in C9 expression.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin CFB expression, e.g., a complement component-associated disease,e.g., PNH, aHUS, or rheumatoid arthritis. The methods includeadministering to the subject a therapeutically effective amount of theiRNA agent, e.g., dsRNA, or vector of the invention, thereby preventingat least one symptom in the subject having a disorder that would benefitfrom reduction in CFB expression. For example, the invention providesmethods for preventing hemolysis in a subject suffering from a disorderthat would benefit from reduction in CFB expression, e.g., a complementcomponent-associated disease, e.g., PNH, aHUS, or rheumatoid arthritis.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin C3 expression, e.g., a complement component-associated disease, e.g.,PNH, aHUS, or rheumatoid arthritis. The methods include administering tothe subject a therapeutically effective amount of the iRNA agent, e.g.,dsRNA, or vector of the invention, thereby preventing at least onesymptom in the subject having a disorder that would benefit fromreduction in C3 expression. For example, the invention provides methodsfor preventing hemolysis in a subject suffering from a disorder thatwould benefit from reduction in C3 expression, e.g., a complementcomponent-associated disease, e.g., PNH, aHUS, or rheumatoid arthritis.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin C9 expression, e.g., a complement component-associated disease, e.g.,PNH, aHUS, or rheumatoid arthritis. The methods include administering tothe subject a therapeutically effective amount of the iRNA agent, e.g.,dsRNA, or vector of the invention, thereby preventing at least onesymptom in the subject having a disorder that would benefit fromreduction in C9 expression. For example, the invention provides methodsfor preventing hemolysis in a subject suffering from a disorder thatwould benefit from reduction in C9 expression, e.g., a complementcomponent-associated disease, e.g., PNH, aHUS, or rheumatoid arthritis.

In another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of CFB expression.

In a further aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of C3 expression.

In yet another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of C9 expression.

In yet another aspect, the present invention provides use of an iRNAagent, e.g., a dsRNA, of the invention targeting a CFB gene or apharmaceutical composition comprising an iRNA agent targeting a CFB genein the manufacture of a medicament for treating a subject, e.g., asubject that would benefit from a reduction and/or inhibition of CFBexpression, such as a subject having a disorder that would benefit fromreduction in CFB expression, e.g., a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis.

In a further aspect, the present invention provides use of an iRNAagent, e.g., a dsRNA, of the invention targeting a C3 gene or apharmaceutical composition comprising an iRNA agent targeting a C3 genein the manufacture of a medicament for treating a subject, e.g., asubject that would benefit from a reduction and/or inhibition of C3expression, such as a subject having a disorder that would benefit fromreduction in C3 expression, e.g., a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis.

In yet a further aspect, the present invention provides use of an iRNAagent, e.g., a dsRNA, of the invention targeting a C9 gene or apharmaceutical composition comprising an iRNA agent targeting a C9 genein the manufacture of a medicament for treating a subject, e.g., asubject that would benefit from a reduction and/or inhibition of C9expression, such as a subject having a disorder that would benefit fromreduction in C9 expression, e.g., a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis.

In another aspect, the invention provides uses of an iRNA, e.g., adsRNA, of the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of CFB expression, such as a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis.

In another aspect, the invention provides uses of an iRNA, e.g., adsRNA, of the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of C3 expression, such as a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis.

In another aspect, the invention provides uses of an iRNA, e.g., adsRNA, of the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of C9 expression, such as a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for preventingat least one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of CFB expression, such as acomplement component-associated disease, e.g., PNH, aHUS, or rheumatoidarthritis.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for preventingat least one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of C3 expression, such as acomplement component-associated disease, e.g., PNH, aHUS, or rheumatoidarthritis.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for preventingat least one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of C9 expression, such as acomplement component-associated disease, e.g., PNH, aHUS, or rheumatoidarthritis.

In some aspects of the invention, the methods which include either asingle iRNA agent of the invention or a combination of iRNA agents,further include administering to the subject one or more additionaltherapeutic agents.

In some aspects, the additional therapeutic agent is an iRNA agenttargeting a C5 gene, such as described in U.S. Provisional PatentApplication No. 61/782,531, filed on Mar. 14, 2013, U.S. ProvisionalPatent Application Ser. No. 61/837,3991, filed on Jun. 20, 2013, andU.S. Provisional Patent Application Ser. No. 61/904,579, filed on Nov.15, 2013, the entire contents of each of which are hereby incorporatedherein by reference.

In other aspects, the additional therapeutic agent is an anti-complementcomponent C5 antibody, or antigen-binding fragment thereof (e.g.,eculizumab). Eculizumab is a humanized monoclonal IgG2/4, kappa lightchain antibody that specifically binds complement component C5 with highaffinity and inhibits cleavage of C5 to C5a and C5b, thereby inhibitingthe generation of the terminal complement complex C5b-9. Eculizumab isdescribed in U.S. Pat. No. 6,355,245, the entire contents of which areincorporated herein by reference.

In yet other aspects, the additional therapeutic is a C3 peptideinhibitor, or analog thereof. In one embodiment, the C3 peptideinhibitor is compstatin. Compstatin is a cyclic tridecapeptide withpotent and selective C3 inhibitory activity. Compstatin, and itsanalogs, are described in U.S. Pat. Nos. 7,888,323, 7,989,589, and8,442,776, in U.S. Patent Publication No. 2012/0178694 and 2013/0053302,and in PCT Publication Nos. WO 2012/174055, WO 2012/2178083, WO2013/036778, the entire contents of each of which are incorporatedherein by reference.

Accordingly, in one aspect, the present invention provides methods oftreating a subject having a disorder that would benefit from reductionin CFB expression, e.g., a complement component-associated disease,e.g., PNH, aHUS, or rheumatoid arthritis, which include administering tothe subject, e.g., a human, a therapeutically effective amount of aniRNA agent targeting a CFB gene or a pharmaceutical compositioncomprising an iRNA agent targeting a CFB gene, and an additionaltherapeutic agent, such as an anti-complement component C5 antibody, orantigen-binding fragment thereof (e.g., eculizumab), an iRNA agenttargeting complement component C5, and/or a C3 peptide inhibitor (e.g.,compstatin), thereby treating the subject having a disorder that wouldbenefit from reduction in CFB expression.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in C3expression, e.g., a complement component-associated disease, e.g., PNH,aHUS, or rheumatoid arthritis, which include administering to thesubject, e.g., a human, a therapeutically effective amount of an iRNAagent targeting a C3 gene or a pharmaceutical composition comprising aniRNA agent targeting a C3 gene, and an additional therapeutic agent,such as an anti-complement component C5 antibody, or antigen-bindingfragment thereof (e.g., eculizumab), an iRNA agent targeting complementcomponent C5, and/or a C3 peptide inhibitor (e.g., compstatin), therebytreating the subject having a disorder that would benefit from reductionin C3 expression.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in C9expression, e.g., a complement component-associated disease, e.g., PNH,aHUS, or rheumatoid arthritis, which include administering to thesubject, e.g., a human, a therapeutically effective amount of an iRNAagent targeting a C9 gene or a pharmaceutical composition comprising aniRNA agent targeting a C9 gene, and an additional therapeutic agent,such as an anti-complement component C5 antibody, or antigen-bindingfragment thereof (e.g., eculizumab), an iRNA agent targeting complementcomponent C5, and/or a C3 peptide inhibitor (e.g., compstatin), therebytreating the subject having a disorder that would benefit from reductionin C9 expression.

In another aspect, the invention provides methods of preventing at leastone symptom in a subject having a disorder that would benefit fromreduction in CFB expression, e.g., a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis. The methods includeadministering to the subject a therapeutically effective amount of theiRNA agent, e.g., dsRNA, or vector of the invention, and an additionaltherapeutic agent, such as an anti-complement component C5 antibody, orantigen-binding fragment thereof (e.g., eculizumab), an iRNA agenttargeting complement component C5, and/or a C3 peptide inhibitor (e.g.,compstatin), thereby preventing at least one symptom in the subjecthaving a disorder that would benefit from reduction in CFB expression.

In another aspect, the invention provides methods of preventing at leastone symptom in a subject having a disorder that would benefit fromreduction in C3 expression, e.g., a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis. The methods includeadministering to the subject a therapeutically effective amount of theiRNA agent, e.g., dsRNA, or vector of the invention, and an additionaltherapeutic agent, such as an anti-complement component C5 antibody, orantigen-binding fragment thereof (e.g., eculizumab), an iRNA agenttargeting complement component C5, and/or a C3 peptide inhibitor (e.g.,compstatin), thereby preventing at least one symptom in the subjecthaving a disorder that would benefit from reduction in C3 expression.

In another aspect, the invention provides methods of preventing at leastone symptom in a subject having a disorder that would benefit fromreduction in C9 expression, e.g., a complement component-associateddisease, e.g., PNH, aHUS, or rheumatoid arthritis. The methods includeadministering to the subject a therapeutically effective amount of theiRNA agent, e.g., dsRNA, or vector of the invention, and an additionaltherapeutic agent, such as an anti-complement component C5 antibody, orantigen-binding fragment thereof (e.g., eculizumab), an iRNA agenttargeting complement component C5, and/or a C3 peptide inhibitor (e.g.,compstatin), thereby preventing at least one symptom in the subjecthaving a disorder that would benefit from reduction in C9 expression.

In another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention andan additional therapeutic agent, such as an anti-complement component C5antibody, or antigen-binding fragment thereof (e.g., eculizumab), aniRNA agent targeting complement component C5, and/or a C3 peptideinhibitor (e.g., comstatin), for treating a subject, e.g., a subjectthat would benefit from a reduction and/or inhibition of CFB expression.

In another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention andan additional therapeutic agent, such as an anti-complement component C5antibody, or antigen-binding fragment thereof (e.g., eculizumab), aniRNA agent targeting complement component C5, and/or a C3 peptideinhibitor (e.g., compstatin), for treating a subject, e.g., a subjectthat would benefit from a reduction and/or inhibition of C3 expression.

In another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention andan additional therapeutic agent, such as an anti-complement component C5antibody, or antigen-binding fragment thereof (e.g., eculizumab), aniRNA agent targeting complement component C5, and/or a C3 peptideinhibitor (e.g., compstatin), for treating a subject, e.g., a subjectthat would benefit from a reduction and/or inhibition of C9 expression.

In another aspect, the present invention provides uses of an iRNA agent,e.g., a dsRNA, of the invention targeting a CFB gene or a pharmaceuticalcomposition comprising an iRNA agent targeting a CFB gene in themanufacture of a medicament for use in combination with an additionaltherapeutic agent, such as an anti-complement component CFB antibody, orantigen-binding fragment thereof (e.g., eculizumab), an iRNA agenttargeting complement component C5, and/or a C3 peptide inhibitor (e.g.,compstatin), for treating a subject, e.g., a subject that would benefitfrom a reduction and/or inhibition of CFB expression, e.g., a complementcomponent-associated disease, e.g., PNH, aHUS, or rheumatoid arthritis.

In another aspect, the present invention provides uses of an iRNA agent,e.g., a dsRNA, of the invention targeting a C3 gene or a pharmaceuticalcomposition comprising an iRNA agent targeting a C3 gene in themanufacture of a medicament for use in combination with an additionaltherapeutic agent, such as an anti-complement component C3 antibody, orantigen-binding fragment thereof (e.g., eculizumab), an iRNA agenttargeting complement component C5, and/or a C3 peptide inhibitor (e.g.,compstatin), for treating a subject, e.g., a subject that would benefitfrom a reduction and/or inhibition of C3 expression, e.g., a complementcomponent-associated disease, e.g., PNH, aHUS, or rheumatoid arthritis.

In another aspect, the present invention provides uses of an iRNA agent,e.g., a dsRNA, of the invention targeting a C9 gene or a pharmaceuticalcomposition comprising an iRNA agent targeting a C9 gene in themanufacture of a medicament for use in combination with an additionaltherapeutic agent, such as an anti-complement component C9 antibody, orantigen-binding fragment thereof (e.g., eculizumab), an iRNA agenttargeting complement component C5, and/or a C3 peptide inhibitor (e.g.,compstatin), for treating a subject, e.g., a subject that would benefitfrom a reduction and/or inhibition of C9 expression, e.g., a complementcomponent-associated disease, e.g., PNH, aHUS, or rheumatoid arthritis.

In yet another aspect, the invention provides uses of an iRNA agent,e.g., a dsRNA, of the invention, and an additional therapeutic agent,such as an anti-complement component C5 antibody, or antigen-bindingfragment thereof (e.g., eculizumab), an iRNA agent targeting complementcomponent C5, and/or a C3 peptide inhibitor (e.g., compstatin), forpreventing at least one symptom in a subject suffering from a disorderthat would benefit from a reduction and/or inhibition of CFB expression,such as a complement component-associated disease, e.g., PNH, aHUS, orrheumatoid arthritis.

In yet another aspect, the invention provides uses of an iRNA agent,e.g., a dsRNA, of the invention, and an additional therapeutic agent,such as an anti-complement component C5 antibody, or antigen-bindingfragment thereof (e.g., eculizumab), an iRNA agent targeting complementcomponent C5, and/or a C3 peptide inhibitor (e.g., compstatin), forpreventing at least one symptom in a subject suffering from a disorderthat would benefit from a reduction and/or inhibition of C3 expression,such as a complement component-associated disease, e.g., PNH, aHUS, orrheumatoid arthritis.

In yet another aspect, the invention provides uses of an iRNA agent,e.g., a dsRNA, of the invention, and an additional therapeutic agent,such as an anti-complement component C5 antibody, or antigen-bindingfragment thereof (e.g., eculizumab), an iRNA agent targeting complementcomponent C5, and/or a C3 peptide inhibitor (e.g., compstatin), forpreventing at least one symptom in a subject suffering from a disorderthat would benefit from a reduction and/or inhibition of C9 expression,such as a complement component-associated disease, e.g., PNH, aHUS, orrheumatoid arthritis.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for use incombination with an additional therapeutic agent, such as ananti-complement component C5 antibody, or antigen-binding fragmentthereof (e.g., eculizumab), an iRNA agent targeting complement componentC5, and/or a C3 peptide inhibitor (e.g., compstatin), for preventing atleast one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of CFB expression, such as acomplement component-associated disease, e.g., PNH, aHUS, or rheumatoidarthritis.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for use incombination with an additional therapeutic agent, such as ananti-complement component C5 antibody, or antigen-binding fragmentthereof (e.g., eculizumab), an iRNA agent targeting complement componentC5, and/or a C3 peptide inhibitor (e.g., compstatin), for preventing atleast one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of C3 expression, such as acomplement component-associated disease, e.g., PNH, aHUS, or rheumatoidarthritis.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for use incombination with an additional therapeutic agent, such as ananti-complement component C5 antibody, or antigen-binding fragmentthereof (e.g., eculizumab), an iRNA agent targeting complement componentC5, and/or a C3 peptide inhibitor (e.g., compstatin), for preventing atleast one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of C9 expression, such as acomplement component-associated disease, e.g., PNH, aHUS, or rheumatoidarthritis.

In one embodiment, an iRNA agent targeting CFB, C3, or C9 isadministered to a subject having a complement component-associateddisease as described herein such that CFB, C3, and/or C9 levels, e.g.,in a cell, tissue, blood, urine or other tissue or fluid of the subjectare reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about99% or more and, subsequently, an additional therapeutic is administeredto the subject.

The additional therapeutic may be an anti-complement component C5antibody, or antigen-binding fragment or derivative thereof. In oneembodiment, the anti-complement component C5 antibody is eculizumab(SOLIRIS®), or antigen-binding fragment or derivative thereof.

The methods of the invention comprising administration of an iRNA agentof the invention and eculizumab to a subject may further compriseadministration of a meningococcal vaccine to the subject.

The additional therapeutic, e.g., eculizumab and/or a meningococcalvaccine, may be administered to the subject at the same time as the iRNAagent targeting CFB, C3, and/or C9 (and/or C5) or at a different time.

Moreover, the additional therapeutic, e.g., eculizumab, may beadministered to the subject in the same formulation as the iRNA agenttargeting CFB, C3, and/or C9 (and/or C5) or in a different formulationas the iRNA agent targeting CFB, C3, and/or C9 (and/or C5).

Eculizumab dosage regimens are described in, for example, the productinsert for eculizumab (SOLIRIS®) and in U.S. Patent Application No.2012/0225056, the entire contents of each of which are incorporatedherein by reference. In exemplary methods of the invention for treatinga complement component-associated disease, e.g., PNH, aHUS, orrheumatoid arthritis, an iRNA agent targeting, e.g., CFB, C3, or C9, isadministered (e.g., subcutaneously) to the subject first, such that theC5 levels in the subject are reduced (e.g., by at least about 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more) andsubsequently eculizumab is administered at doses lower than the onesdescribed in the product insert for SOLIRIS®. For example, eculizumabmay be administered to the subject weekly at a dose less than about 600mg for 4 weeks followed by a fifth dose at about one week later of lessthan about 900 mg, followed by a dose less than about 900 mg about everytwo weeks thereafter. Eculizumab may also be administered to the subjectweekly at a dose less than about 900 mg for 4 weeks followed by a fifthdose at about one week later of less than about 1200 mg, followed by adose less than about 1200 mg about every two weeks thereafter. If thesubject is less than 18 years of age, eculizumab may be administered tothe subject weekly at a dose less than about 900 mg for 4 weeks followedby a fifth dose at about one week later of less than about 1200 mg,followed by a dose less than about 1200 mg about every two weeksthereafter; or if the subject is less than 18 years of age, eculizumabmay be administered to the subject weekly at a dose less than about 600mg for 2 weeks followed by a third dose at about one week later of lessthan about 900 mg, followed by a dose less than about 900 mg about everytwo weeks thereafter; or if the subject is less than 18 years of age,eculizumab may be administered to the subject weekly at a dose less thanabout 600 mg for 2 weeks followed by a third dose at about one weeklater of less than about 600 mg, followed by a dose less than about 600mg about every two weeks thereafter; or if the subject is less than 18years of age, eculizumab may be administered to the subject weekly at adose less than about 600 mg for 1 week followed by a second dose atabout one week later of less than about 300 mg, followed by a dose lessthan about 300 mg about every two weeks thereafter; or if the subject isless than 18 years of age, eculizumab may be administered to the subjectweekly at a dose less than about 300 mg for 1 week followed by a seconddose at about one week later of less than about 300 mg, followed by adose less than about 300 mg about every two weeks thereafter. If thesubject is receiving plamapheresis or plasma exchange, eculizumab may beadministered to the subject at a dose less than about 300 mg (e.g., ifthe most recent does of eculizumab was about 300 mg) or less than about600 mg (e.g., if the most recent does of eculizumab was about 600 mg ormore). If the subject is receiving plasma infusion, eculizumab may beadministered to the subject at a dose less than about 300 mg (e.g., ifthe most recent does of eculizumab was about 300 mg or more). The lowerdoses of eculizumab allow for either subcutaneous or intravenousadministration of eculizumab.

In the combination therapies of the present invention comprisingeculizumab, eculizumab may be administered to the subject, e.g.,subcutaneously, at a dose of about 0.01 mg/kg to about 10 mg/kg, orabout 5 mg/kg to about 10 mg/kg, or about 0.5 mg/kg to about 15 mg/kg.For example, eculizumab may be administered to the subject, e.g.,subcutaneously, at a dose of 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5mg/kg, 10 mg/kg, 10.5 mg/kg, 11 mg/kg, 11.5 mg/kg, 12 mg/kg, 12.5 mg/kg,13 mg/kg, 13.5 mg/kg, 14 mg/kg, 14.5 mg/kg, or 15 mg/kg.

The methods and uses of the invention include administering acomposition described herein such that expression of the target CFB, C3,and/or C9 (and/or C5) gene is decreased, such as for about 1, 2, 3, 4,5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68,72, 76, or about 80 hours. In one embodiment, expression of the targetgene is decreased for an extended duration, e.g., at least about two,three, four, five, six, seven days or more, e.g., about one week, twoweeks, three weeks, or about four weeks or longer.

Administration of the dsRNA according to the methods and uses of theinvention may result in a reduction of the severity, signs, symptoms,and/or markers of such diseases or disorders in a patient with acomplement component-associated disease. By “reduction” in this contextis meant a statistically significant decrease in such level. Thereduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of a hemolytic disorder may be assessed,for example, by periodic monitoring of LDH and CH₅₀ levels. Comparisonsof the later readings with the initial readings provide a physician anindication of whether the treatment is effective. It is well within theability of one skilled in the art to monitor efficacy of treatment orprevention by measuring any one of such parameters, or any combinationof parameters. In connection with the administration of an iRNAtargeting CFB, C3, and/or C9, or pharmaceutical composition thereof,“effective against” a complement component-associated disease indicatesthat administration in a clinically appropriate manner results in abeneficial effect for at least a statistically significant fraction ofpatients, such as improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating a complement component-associated disease and the relatedcauses.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the Rheumatoid Arthritis Severity Scale (RASS). Any positivechange resulting in e.g., lessening of severity of disease measuredusing the appropriate scale, represents adequate treatment using an iRNAor iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg,0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg,0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg,0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg,1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kgdsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kgdsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kgdsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kgdsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kgdsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kgdsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kgdsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kgdsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kgdsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kgdsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kgdsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kgdsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kgdsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kgdsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kgdsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kgdsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kgdsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kgdsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kgdsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA,30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about50 mg/kg dsRNA. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of iRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment,when a composition of the invention comprises a dsRNA as describedherein and an N-acetylgalactosamine, subjects can be administered atherapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes a modification (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides), including one such motif at or near the cleavage site ofthe agent, six phosphorothioate linkages, and a ligand, such an agent isadministered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 toabout 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/mg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The iRNA can be administered by intravenous infusion over a period oftime, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or about a 25 minute period. The administrationmay be repeated, for example, on a regular basis, such as weekly,biweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration weekly or biweekly for three months, administrationcan be repeated once per month, for six months or a year or longer.

Administration of the iRNA can reduce CFB, C3, and/or C9 (and/or C5)levels, e.g., in a cell, tissue, blood, urine or other compartment ofthe patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion, and monitored foradverse effects, such as an allergic reaction. In another example, thepatient can be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Owing to the inhibitory effects on CFB, C3, and/or C9 expression, acomposition according to the invention or a pharmaceutical compositionprepared therefrom can enhance the quality of life.

An iRNA of the invention may be administered in “naked” form, or as a“free iRNA.” A naked iRNA is administered in the absence of apharmaceutical composition. The naked iRNA may be in a suitable buffersolution. The buffer solution may comprise acetate, citrate, prolamine,carbonate, or phosphate, or any combination thereof. In one embodiment,the buffer solution is phosphate buffered saline (PBS). The pH andosmolarity of the buffer solution containing the iRNA can be adjustedsuch that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of CFB,C3, and/or C9 gene expression are those having a complementcomponent-associated disease or disorder as described herein. In oneembodiment, a subject having a complement component-associated diseasehas paroxysmal nocturnal hemoglobinuria (PNH). In another embodiment, asubject having a complement component-associated disease has asthma. Inanother embodiment, a subject having a complement component-associateddisease has rheumatoid arthritis. In yet another embodiment, a subjecthaving a complement component-associated disease has systemic lupuserythmatosis. In one embodiment, a subject having a complementcomponent-associated disease has glomerulonephritis. In anotherembodiment, a subject having a complement component-associated diseasehas psoriasis. In yet another embodiment, a subject having a complementcomponent-associated disease has dermatomyositis bullous pemphigoid. Inone embodiment, a subject having a complement component-associateddisease has atypical hemolytic uremic syndrome. In another embodiment, asubject having a complement component-associated disease has Shiga toxinE. coli-related hemolytic uremic syndrome. In another embodiment, asubject having a complement component-associated disease has myastheniagravis. In yet another embodiment, a subject having a complementcomponent-associated disease has neuromyelistis optica. In oneembodiment, a subject having a complement component-associated diseasehas dense deposit disease. In one embodiment, a subject having acomplement component-associated disease has C3 neuropathy. In anotherembodiment, a subject having a complement component-associated diseasehas age-related macular degeneration. In another embodiment, a subjecthaving a complement component-associated disease has cold agglutinindisease. In one embodiment, a subject having a complementcomponent-associated disease has anti-neutrophil cytoplasmicantibody-associated vasculitis. In another embodiment, a subject havinga complement component-associated disease has humoral and vasculartransplant rejection. In one embodiment, a subject having a complementcomponent-associated disease has graft dysfunction. In one embodiment, asubject having a complement component-associated disease has had amyocardial infarction. In another embodiment, a subject having acomplement component-associated disease is a sensitized recipient of atransplant. In yet another embodiment, a subject having a complementcomponent-associated disease has sepsis.

Treatment of a subject that would benefit from a reduction and/orinhibition of CFB, C3, and/or C9 gene expression includes therapeuticand prophylactic (e.g., the subject is to undergo sensitized (orallogenic) transplant surgery treatment.

The invention further provides methods and uses of an iRNA agent or apharmaceutical composition thereof (including methods and uses of aniRNA agent or a pharmaceutical composition comprising an iRNA agent andan additional therapeutic agent, e.g. an anti-complement component C5antibody, or antigen-binding fragment thereof) for treating a subjectthat would benefit from reduction and/or inhibition of a target gene ofthe invention, e.g., CFB, C3, and C9, expression, e.g., a subject havinga complement component-associated disease, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders. Forexample, in certain embodiments, an iRNA targeting CFB is administeredin combination with, e.g., an agent useful in treating a complementcomponent-associated disease as described elsewhere herein.

For example, additional therapeutics and therapeutic methods suitablefor treating a subject that would benefit from reduction in CFB, C3,and/or C9 expression, e.g., a subject having a complementcomponent-associated disease, include plasmaphoresis, thrombolytictherapy (e.g., streptokinase), antiplatelet agents, folic acid,corticosteroids; immunosuppressive agents; estrogens, methotrexate,6-MP, azathioprine sulphasalazine, mesalazine, olsalazine,chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate(intramuscular and oral), azathioprine, cochicine, corticosteroids(oral, inhaled and local injection), beta-2 adrenoreceptor agonists(salbutamol, terbutaline, salmeteral), xanthines (theophylline,aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium andoxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines, such asTNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors),IL-1β converting enzyme inhibitors, TNFαconverting enzyme (TACE)inhibitors, T-cell signalling inhibitors, such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG(Lenercept)), sIL-1RI, sIL-1RII, and sIL-6R), antiinflammatory cytokines(e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid,hydroxychloroquine sulfate, rofecoxib, etanercept, infliximonoclonalantibody, naproxen, valdecoxib, sulfasalazine, methylprednisolone,meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin,triamcinolone acetonide, propoxyphene napsylate/apap, folate,nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium,oxaprozin, oxycodone hydrochloride, hydrocodone bitartrate/apap,diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant,tramadol hydrochloride, salsalate, sulindac, cyanocobalamin/folicacid/pyridoxine, acetaminophen, alendronate sodium, prednisolone,morphine sulfate, lidocaine hydrochloride, indomethacin, glucosaminesulf/chondroitin, amitriptyline hydrochloride, sulfadiazine, oxycodonehydrochloride/acetaminophen, olopatadine hydrochloride, misoprostol,naproxen sodium, omeprazole, cyclophosphamide, rituximonoclonalantibody, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15,BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485,CDC-801, Mesopram, cyclosporine, cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Bayer); cA2/infliximonoclonal antibody (chimeric anti-TNFαantibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgGfusion protein; Immunex; see e.g., (1994) Arthr. Rheum. 37: S295; (1996)J. Invest. Med. 44: 235A); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusionprotein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depletingprimatized anti-CD4 antibody; IDEC/SmithKline; see e.g., (1995) Arthr.Rheum. 38: 5185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusionproteins; Seragen; see e.g., (1993) Arthrit. Rheum. 36: 1223); Anti-Tac(humanized anti-IL-2Rα; Protein Design Labs/Roche); IL-4(anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4;IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1receptor antagonist; Synergen/Amgen); anakinra (Kineret®/Amgen);TNF-bp/s-TNF (soluble TNF binding protein; see e.g., (1996) Arthr.Rheum. 39(9 (supplement)): 5284; (1995) Amer. J. Physiol.—Heart andCirc. Physiol. 268: 37-42); R973401 (phosphodiesterase Type IVinhibitor; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): 5282);MK-966 (COX-2 Inhibitor; see e.g., (1996) Arthr. Rheum. 39(9(supplement): S81); Iloprost (see e.g., (1996) Arthr. Rheum. 39(9(supplement): S82); methotrexate; thalidomide (see e.g., (1996) Arthr.Rheum. 39(9 (supplement): 5282) and thalidomide-related drugs (e.g.,Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; seee.g., (1996) Arthr. Rheum. 39(9 (supplement): 5131; (1996) Inflamm. Res.45: 103-107); tranexamic acid (inhibitor of plasminogen activation; seee.g., (1996) Arthr. Rheum. 39(9 (supplement): 5284); T-614 (cytokineinhibitor; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): 5282);prostaglandin E1 (see e.g., (1996) Arthr. Rheum. 39(9 (supplement):5282); Tenidap (non-steroidal anti-inflammatory drug; see e.g., (1996)Arthr. Rheum. 39(9 (supplement): 5280); Naproxen (non-steroidalanti-inflammatory drug; see e.g., (1996) Neuro. Report 7: 1209-1213);Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen(non-steroidal anti-inflammatory drug); Piroxicam (non-steroidalanti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatorydrug); Indomethacin (non-steroidal anti-inflammatory drug);Sulfasalazine (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): 5281);Azathioprine (see e.g., (1996) Arthr. Rheum. 39(9 (supplement): 5281);ICE inhibitor (inhibitor of the enzyme interleukin-1β convertingenzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinasezap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitors ofvascular endothelial cell growth factor or vascular endothelial cellgrowth factor receptor; inhibitors of angiogenesis); corticosteroidanti-inflammatory drugs (e.g., SB203580); TNF-convertase inhibitors;anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11 (see e.g.,(1996) Arthr. Rheum. 39(9 (supplement): 5296); interleukin-13 (see e.g.,(1996) Arthr. Rheum. 39(9 (supplement): 5308); interleukin-17 inhibitors(see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S120); gold;penicillamine; chloroquine; chlorambucil; hydroxychloroquine;cyclosporine; cyclophosphamide; total lymphoid irradiation;anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally-administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals,Inc.); ICAM-1 antisense phosphorothioate oligo-deoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995)Rheum. Dis. Clin. North Am. 21: 759-777); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506);sirolimus (rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko,M. et al. (2007) J. Med. Chem. 50(4): 641-662); antivirals andimmune-modulating agents, small molecule inhibitor of KDR, smallmolecule inhibitor of Tie-2; methotrexate; prednisone; celecoxib; folicacid; hydroxychloroquine sulfate; rofecoxib; etanercept;infliximonoclonal antibody; leflunomide; naproxen; valdecoxib;sulfasalazine; methylprednisolone; ibuprofen; meloxicam;methylprednisolone acetate; gold sodium thiomalate; aspirin;azathioprine; triamcinolone acetonide; propxyphene napsylate/apap;folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium;oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenacsodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl;salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen;alendronate sodium; prednisolone; morphine sulfate; lidocainehydrochloride; indomethacin; glucosamine sulfate/chondroitin;cyclosporine; amitriptyline hydrochloride; sulfadiazine; oxycodonehcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium;omeprazole; mycophenolate mofetil; cyclophosphamide; rituximonoclonalantibody; IL-1 TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18;anti-IL 15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast;IC-485; CDC-801; mesopram, albuterol, salmeterol/fluticasone,montelukast sodium, fluticasone propionate, budesonide, prednisone,salmeterol xinafoate, levalbuterol hcl, albuterol sulfate/ipratropium,prednisolone sodium phosphate, triamcinolone acetonide, beclomethasonedipropionate, ipratropium bromide, azithromycin, pirbuterol acetate,prednisolone, theophylline anhydrous, methylprednisolone sodiumsuccinate, clarithromycin, zafirlukast, formoterol fumarate, influenzavirus vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide,allergy injection, cromolyn sodium, fexofenadine hydrochloride,flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhalerassist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacinhcl, doxycycline hyclate, guaifenesin/d-methorphan,p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride,mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin,pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed,phenylephrine/cod/promethazine, codeine/promethazine, cefprozil,dexamethasone, guaifenesin/pseudoephedrine,chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate,epinephrine, methylprednisolone, metaproterenol sulfate, aspirin,nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium,clopidogrel bisulfate, carvedilol, atenolol, morphine sulfate,metoprolol succinate, warfarin sodium, lisinopril, isosorbidemononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase,enalapril maleate, torsemide, retavase, losartan potassium, quinaprilhcl/mag carb, bumetanide, alteplase, enalaprilat, amiodaronehydrochloride, tirofiban hcl m-hydrate, diltiazem hydrochloride,captopril, irbesartan, valsartan, propranolol hydrochloride, fosinoprilsodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium,atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalolhydrochloride, potassium chloride, docusate sodium, dobutamine hcl,alprazolam, pravastatin sodium, atorvastatin calcium, midazolamhydrochloride, meperidine hydrochloride, isosorbide dinitrate,epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,ezetimibe/simvastatin, avasimibe, and cariporide.

The iRNA agent (and/or an anti-complement component C5 antibody) and anadditional therapeutic agent and/or treatment may be administered at thesame time and/or in the same combination, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or at separate times and/or by another method known in theart or described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Transcripts

siRNA design was carried out to identify siRNAs targeting human (Homosapiens), cynomolgus monkey (Macaca fascicularis; henceforth “cyno”),mouse (Mus musculus), and rat (Rattus norvegicus) transcripts. Ingeneral, the design of duplexes used human, mouse, and rat transcriptsfrom the NCBI RefSeq collection, annotated in the NCBI Gene database(http://www.ncbi.nlm.nih.gov/gene/). For cyno, the design used eithertranscripts downloaded from the M. fascicularis genome project(http://macaque.genomics.org.cn/page/species/download.jsp) and/ortranscripts obtained from a liver-derived cDNA library.

Design of CFB siRNAs used the following transcripts from the NCBI RefSeqcollection: Human—NM_001710; Cyno (from the M. fascicularis genomeproject)—ENSMMUP00000000985 (locus=scaffold3881:47830:53620);Mouse—NM_001142706 and NM_008198; and Rat—NM_212466.3.

Design of C3 siRNAs used the following transcripts from the NCBI RefSeqcollection: Human—NM_000064; Cyno (from the M. fascicularis genomeproject)—ENSP00000245907 (locus=chr19:6921416:6963034); Mouse—NM_009778;and Rat—NM_016994

Design of C9 siRNAs used the following transcripts from the NCBI RefSeqcollection: Human—NM_001737; Cyno (from liver cDNA library)—isotig05361;Mouse—NM_013485; AND Rat—NM_057146.

siRNA duplexes were designed in several separate batches, including butnot limited to batches containing duplexes matching human transcriptsonly; human and cyno transcripts; human, cyno, and mouse transcripts;AND human, cyno, mouse, and rat transcripts. Most siRNA duplexes weredesigned that shared 100% identity with the listed human transcript andother species transcripts considered in each design batch (above). Insome instances, however, when the antisense strand:target mRNAcomplementary basepair was a GC or CG pair, siRNA duplexes were designedwith mismatches between duplex and mRNA target at the first antisense(last sense) position (see, e.g. Table 5, oligos with label G21U, G21A,C21A, G21A). In these cases, duplexes were designed with UA or AUbasepairs at the first antisense:last sense pair. Thus the duplexesmaintained complementarity but were mismatched with respect to target(U:C, U:G, A:C, or A:G).

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were then selected that lacked repeats longerthan 7 nucleotides.

The following sets of candidate siRNAs were used in comprehensivesearches against the appropriate transcriptomes (defined as the set ofNM_ and XM_records within the human, mouse, or rat NCBI Refseq sets, andthe cyno transcriptome set in NCBI nucleotide) using an exhaustive“brute-force” algorithm implemented in the python script‘BruteForce.py’.

C3: 46 human/cyno/mouse/rat, 80 human/cyno/mouse, 2384 human/cyno.

C9: 7 human/cyno/mouse/rat, 12 human/cyno/mouse, 816 human/cyno.

CFB: 23 human/cyno/mouse, 1232 human/cyno.

The script next parsed the transcript-oligo alignments to generate ascore based on the position and number of mismatches between the siRNAand any potential ‘off-target’ transcript. The off-target score isweighted to emphasize differences in the ‘seed’ region of siRNAs, inpositions 2-9 from the 5′-end of the molecule.

Each oligo-transcript pair from the brute-force search was given amismatch score by summing the individual mismatch scores; mismatches inthe position 2-9 were counted as 2.8, mismatches in the cleavage sitepositions 10-11 were counted as 1.2, and mismatches in region 12-19counted as 1.0. An additional off-target prediction was carried out bycomparing the frequency of heptamers and octomers derived from 3distinct, seed-derived hexamers of each oligo. The hexamers frompositions 2-7 relative to the 5′ start were used to create 2 heptamersand one octamer. ‘Heptamer1’ was created by adding a 3′-A to thehexamer; heptamer2 was created by adding a 5′-A to the hexamer; theoctomer was created by adding an A to both 5′- and 3′-ends of thehexamer. The frequency of octamers and heptamers in the human, rhesus,mouse, or rat 3′-UTRome (defined as the subsequence of the transcriptomefrom NCBI's Refseq database where the end of the coding region, the‘CDS’, is clearly defined) was pre-calculated. The octamer frequency wasnormalized to the heptamer frequency using the median value from therange of octamer frequencies. A ‘mirSeedScore’ was then calculated bycalculating the sum of ((3×normalized octamer count)+(2×heptamer2count)+(1×heptamer1 count)).

Both siRNAs strands were assigned to a category of specificity accordingto the calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.The duplexes were sorted by the specificity of the antisense strand andthose duplexes whose antisense oligos lacked GC at the first position,lacked G at both positions 13 and 14, and had 3 or more Us or As in theseed region were selected.

For GalNaC-conjugated duplexes, sense 21mer and antisense 23mer oligoswere designed by extending antisense 19mers (described above) to 23nucleotides of target-complementary sequence. All species transcriptsincluded in the design batch were checked for complementarity. Only23mers that preserved 100% sequence complementarity in at least 2species were used. For each duplex, the sense 21mer was specified as thereverse complement of the first 21 nucleotides of the antisense strand.

siRNA Sequence Selection

The following 21/23mer duplex sets for GalNac conjugate design weresynthesized and formed into duplexes.

C3: twenty sense and 20 antisense derived human/cyno/mouse/rat oligopairs, including 6 where the first antisense position was swapped to UA(above); 10 sense and 10 antisense derived human/cyno/mouse oligo pairs,including 3 where the first antisense position was swapped to UA(above); 12 sense and 12 antisense derived human/cyno oligo pairs.

C9: one sense and 1 antisense derived human/cyno/mouse/rat oligo pair; 2sense and 2 antisense derived human/cyno/mouse oligo pairs; 1 sense and1 antisense derived human/cyno/rat oligo pairs; 19 sense and 19antisense derived human/cyno oligo pairs.

CFB: nine sense and 9 antisense derived human/cyno/mouse oligo pairs,including 4 where the first antisense position was swapped to UA(above); 23 sense and 23 antisense derived human/cyno oligo pairs.

A detailed list of CFB sense and antisense strand sequences is shown inTables 3-4.

A detailed list of C3 sense and antisense strand sequences is shown inTables 5-6.

A detailed list of C9 sense and antisense strand sequences is shown inTables 7-8.

siRNA Synthesis

General Small and Medium Scale RNA Synthesis Procedure

RNA oligonucleotides were synthesized at scales between 0.2-500 μmolusing commercially available5′-O-(4,4′-dimethoxytrityl)-2′-O-t-butyldimethylsilyl-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditemonomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine and2-N-isobutyrylguanosine and the corresponding 2′-O-methyl and 2′-fluorophosphoramidites according to standard solid phase oligonucleotidesynthesis protocols. The amidite solutions were prepared at 0.1-0.15 Mconcentration and 5-ethylthio-1H-tetrazole (0.25-0.6 M in acetonitrile)was used as the activator. Phosphorothioate backbone modifications wereintroduced during synthesis using 0.2 M phenylacetyl disulfide (PADS) inlutidine:acetonitrile (1:1) (v;v) or 0.1 M 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine for the oxidationstep. After completion of synthesis, the sequences were cleaved from thesolid support and deprotected using methylamine followed bytriethylamine.3HF to remove any 2′-O-t-butyldimethylsilyl protectinggroups present.

For synthesis scales between 5-500 μmol and fully 2′ modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides where deprotected using 3:1 (v/v) ethanol andconcentrated (28-32%) aqueous ammonia either at 35° C. 16 h or 55° C.for 5.5 h. Prior to ammonia deprotection the oligonucleotides wheretreated with 0.5 M piperidine in acetonitrile for 20 min on the solidsupport. The crude oligonucleotides were analyzed by LC-MS andanion-exchange HPLC (IEX-HPLC). Purification of the oligonucleotides wascarried out by IEX HPLC using: 20 mM phosphate, 10%-15% ACN, pH=8.5(buffer A) and 20 mM phosphate, 10%-15% ACN, 1 M NaBr, pH=8.5 (bufferB). Fractions were analyzed for purity by analytical HPLC. Theproduct-containing fractions with suitable purity were pooled andconcentrated on a rotary evaporator prior to desalting. The samples weredesalted by size exclusion chromatography and lyophilized to dryness.Equal molar amounts of sense and antisense strands were annealed in1×PBS buffer to prepare the corresponding siRNA duplexes.

For small scales (0.2-1 μmol), synthesis was performed on a MerMade 192synthesizer in a 96 well format. In case of fully 2′-modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides where deprotected using methylamine at room temperaturefor 30-60 min followed by incubation at 60° C. for 30 min or using 3:1(v/v) ethanol and concentrated (28-32%) aqueous ammonia at roomtemperature for 30-60 min followed by incubation at 40° C. for 1.5hours. The crude oligonucleotides were then precipitated in a solutionof acetonitrile:acetone (9:1) and isolated by centrifugation anddecanting the supernatant. The crude oligonucleotide pellet wasre-suspended in 20 mM NaOAc buffer and analyzed by LC-MS and anionexchange HPLC. The crude oligonucleotide sequences were desalted in 96deep well plates on a 5 mL HiTrap Sephadex G25 column (GE Healthcare).In each well about 1.5 mL samples corresponding to an individualsequence was collected. These purified desalted oligonucleotides wereanalyzed by LC-MS and anion exchange chromatography. Duplexes wereprepared by annealing equimolar amounts of sense and antisense sequenceson a Tecan robot. Concentration of duplexes was adjusted to 10 μM in1×PBS buffer.

I. Synthesis of GalNAc-Conjugated Oligonucleotides for In Vivo Analysis

Oligonucleotides conjugated with GalNAc ligand at their 3′-terminus weresynthesized at scales between 0.2-500 μmol using a solid supportpre-loaded with a Y-shaped linker bearing a 4,4′-dimethoxytrityl(DMT)-protected primary hydroxy group for oligonucleotide synthesis anda GalNAc ligand attached through a tether.

For synthesis of GalNAc conjugates in the scales between 5-500 μmol, theabove synthesis protocol for RNA was followed with the followingadaptions: For polystyrene-based synthesis supports 5% dichloroaceticacid in toluene was used for DMT-cleavage during synthesis. Cleavagefrom the support and deprotection was performed as described above.Phosphorothioate-rich sequences (usually >5 phorphorothioates) weresynthesized without removing the final 5′-DMT group (“DMT-on”) and,after cleavage and deprotection as described above, purified by reversephase HPLC using 50 mM ammonium acetate in water (buffer A) and 50 mMammoniumacetate in 80% acetonitirile (buffer B). Fractions were analyzedfor purity by analytical HPLC and/or LC-MS. The product-containingfractions with suitable purity were pooled and concentrated on a rotaryevaporator. The DMT-group was removed using 20%-25% acetic acid in wateruntil completion. The samples were desalted by size exclusionchromatography and lyophilized to dryness. Equal molar amounts of senseand antisense strands were annealed in 1×PBS buffer to prepare thecorresponding siRNA duplexes.

For small scale synthesis of GalNAc conjugates (0.2-1 μmol), includingsequences with multiple phosphorothioate linkages, the protocolsdescribed above for synthesis of RNA or fully 2′-F/2′-OMe-containingsequences on MerMade platform were applied. Synthesis was performed onpre-packed columns containing GalNAc-functionalized controlled poreglass support.

Example 2. In Vitro Screening Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Cells were washed andre-suspended at 0.25×10⁶ cells/ml. During transfections, cells wereplated onto a 96-well plate with about 20,000 cells per well.

Primary mouse hepatocytes (PMH) were freshly isolated from a C57BL/6female mouse (Charles River Laboratories International, Inc.Willmington, Mass.) less than 1 hour prior to transfections and grown inprimary hepatocyte media. Cells were resuspended at 0.11×10⁶ cells/ml inInVitroGRO CP Rat (plating) medium (Celsis In Vitro Technologies,catalog number S01494). During transfections, cells were plated onto aBD BioCoat 96 well collagen plate (BD, 356407) at 10,000 cells per welland incubated at 37° C. in an atmosphere of 5% CO₂.

For Hep3B and PMH, transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. catalog number 13778-150) to 5 μl of each siRNA duplexto an individual well in a 96-well plate. The mixture was then incubatedat room temperature for 20 minutes. Eighty μl of complete growth mediawithout antibiotic containing the appropriate cell number were thenadded to the siRNA mixture. Cells were incubated for 24 hours prior toRNA purification.

Single dose experiments were performed at 1 nM and 0.01 nM final duplexconcentration for GalNAc modified sequences. Dose response experimentswere done at 3, 1, 0.3, 0.1, 0.037, 0.0123, 0.00412, and 0.00137 nMfinal duplex concentration for primary mouse hepatocytes and at 3, 1,0.3, 0.1, 0.037, 0.0123, 0.00412, 0.00137, 0.00046, 0.00015, 0.00005,and 0.000017 nM final duplex concentration for Hep3B cells.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (themixing speed was the same throughout the process). Ten microliters ofmagnetic beads and 80 μl Lysis/Binding Buffer mixture were added to around bottom plate and mixed for 1 minute. Magnetic beads were capturedusing a magnetic stand and the supernatant was removed withoutdisturbing the beads. After removing the supernatant, the lysed cellswere added to the remaining beads and mixed for 5 minutes. Afterremoving the supernatant, magnetic beads were washed 2 times with 150 μlWash Buffer A and mixed for 1 minute. The beads were captured again andthe supernatant was removed. The beads were then washed with 150 μl WashBuffer B, captured and the supernatant was removed. The beads were nextwashed with 150 μl Elution Buffer, captured and the supernatant removed.Finally, the beads were allowed to dry for 2 minutes. After drying, 50μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Thebeads were captured on magnet for 5 minutes. Forty-five μl ofsupernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction as prepared. Equal volumes master mix and RNA were mixed for afinal volume of 12 μl for in vitro screened or 20 μl for in vivoscreened samples. cDNA was generated using a Bio-Rad C-1000 or S-1000thermal cycler (Hercules, Calif.) through the following steps: 25° C.for 10 minutes, 37° C. for 120 minutes, 85° C. for 5 seconds, and 4° C.hold.

Real Time PCR

Two μl of cDNA were added to a master mix containing 2 μl of H₂O, 0.5 μlGAPDH TaqMan Probe (Life Technologies catalog number 4326317E for Hep3Bcells, catalog number 352339E for primary mouse hepatocytes or customprobe for cynomolgus primary hepatocytes), 0.5 μl of appropriate TaqManprobe (Life Technologies c catalog number Hs00156197_m1 for Hep3B cellsor mm00439275_m1 for Primary Mouse Hepatoctyes or custom probe forcynomolgus primary hepatocytes) and 5 μl Lightcycler 480 probe mastermix (Roche catalog number 04887301001) per well in a 384 well plates(Roche catalog number 04887301001). Real time PCR was performed in anRoche LC480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Forin vitro screening, each duplex was tested with two biologicalreplicates unless otherwise noted and each Real Time PCR was performedin duplicate technical replicates. For in vivo screening, each duplexwas tested in one or more experiments (3 mice per group) and each RealTime PCR was run in duplicate technical replicates.

To calculate relative fold change in mRNA levels, real time data wereanalyzed using the ΔΔCt method and normalized to assays performed withcells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀swere calculated using a 4 parameter fit model using XLFit and normalizedto cells transfected with AD-1955 over the same dose range, or to itsown lowest dose.

The sense and antisense sequences of AD-1955 are:

SENSE: (SEQ ID NO: 39) cuuAcGcuGAGuAcuucGAdTsdT ANTISENSE:(SEQ ID NO: 40) UCGAAGuACUcAGCGuAAGdTsdT.

Table 9 shows the results of a single dose screen in Hep3B cellstransfected with the indicated CFB GalNAC conjugated iRNAs. Data areexpressed as percent of message remaining relative to untreated cells.

Table 10 shows the results of a single dose screen in primary mousehepatocytes transfected with the indicated CFB GalNAC conjugated iRNAs.Data are expressed as percent of message remaining relative to untreatedcells.

Table 11 shows the dose response in Hep3B cells transfected with theindicated CFB GalNAC conjugated iRNAs. The indicated IC₅₀ valuesrepresent the IC₅₀ values relative to untreated cells.

Table 12 shows the dose response in primary mouse hepatocytestransfected with the indicated CFB GalNAC conjugated iRNAs. Theindicated IC₅₀ values represent the IC₅₀ values relative to untreatedcells.

Table 13 shows the results of a single dose screen in primary mousehepatocytes transfected with the indicated C9 GalNAC conjugated iRNAs.Data are expressed as percent of message remaining relative to untreatedcells.

Table 14 shows the results of a single dose screen in primary mousehepatocytes transfected with the indicated C3 GalNAC conjugated iRNAs.Data are expressed as percent of message remaining relative to untreatedcells.

Table 15 shows the results of a single dose screen in Hep3B cellstransfected with the indicated C3 GalNAC conjugated iRNAs. Data areexpressed as percent of message remaining relative to untreated cells.

Table 16 shows the dose response in primary mouse hepatocytestransfected with the indicated C3 GalNAC conjugated iRNAs. The indicatedIC₅₀ values represent the IC₅₀ values relative to untreated cells.

Table 17 shows the dose response in Hep3B cells transfected with theindicated C3 GalNAC conjugated iRNAs. The indicated IC₅₀ valuesrepresent the IC₅₀ values relative to untreated cells.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4- hydroxyprolinolHyp-(GalNAc-alkyl)3

TABLE 3 Complement Factor B (CFB) unmodified sequencesHuman CFB Sequences Sense Sequence Antisense Sequence(SEQ ID NOS: 41-71, (SEQ ID NOS: 72-102, Duplex respectively, inPosition in Antisense respectively, in Position in ID Sense IDorder of appearance) NM_001710.5 ID order of appearance) NM_001710.5 AD-A-122021.1 AUUCCUGAAUUUUAUGACUAU 1987-2007 A-122022.1AUAGUCAUAAAAUUCAGGAAUUC 1985-2007 60315.1 AD- A-122009.1CCUGAUCAAGCUCAAGAAUAA 2016-2036 A-122010.1 UUAUUCUUGAGCUUGAUCAGGGC2014-2036 60326.1 AD- A-122017.1 GAAGCAGGAAUUCCUGAAUUU 1978-1998A-122018.1 AAAUUCAGGAAUUCCUGCUUCUU 1976-1998 60303.1 AD- A-121995.1AGCAACAUGUGUUCAAAGUCA 1628-1648 A-121996.1 UGACUUUGAACACAUGUUGCUCA1626-1648 60331.1 AD- A-122015.1 GCUGUGGUGUCUGAGUACUUU 1822-1842A-122016.1 AAAGUACUCAGACACCACAGCCC 1820-1842 60344.1 AD- A-122031.1AAGUGUCUAGUCAACUUAAUU 1153-1173 A-122032.1 AAUUAAGUUGACUAGACACUUUU1151-1173 60345.1 AD- A-121991.1 AGCUGUGAGAGAGAUGCUCAA 2245-2265A-121992.1 UUGAGCAUCUCUCUCACAGCUGC 2243-2265 60319.1 AD- A-122003.1AGCCAAAAAGUGUCUAGUCAA 1146-1166 A-122004.1 UUGACUAGACACUUUUUGGCUCC1144-1166 60308.1 AD- A-122011.1 UGUGAGUGAUGAGAUCUCUUU 648-668A-122012.1 AAAGAGAUCUCAUCACUCACAUU 646-668 60332.1 AD- A-121989.1AAUUGAGAAGGUGGCAAGUUA 1170-1190 A-121990.1 UAACUUGCCACCUUCUCAAUUAA1168-1190 60313.1 AD- A-122023.1 CAACAUGUGUUCAAAGUCAAG 1630-1650A-122024.1 CUUGACUUUGAACACAUGUUGCU 1628-1650 60321.1 AD- A-122025.1UGUGAGAGAGAUGCUCAAUAU 2248-2268 A-122026.1 AUAUUGAGCAUCUCUCUCACAGC2246-2268 60327.1 AD- A-122001.1 GUCUAGUCAACUUAAUUGAGA 1157-1177A-122002.1 UCUCAAUUAAGUUGACUAGACAC 1155-1177 60302.1 AD- A-121993.1UCCAAGAAAGACAAUGAGCAA 1612-1632 A-121994.1 UUGCUCAUUGUCUUUCUUGGAAG1610-1632 60325.1 AD- A-121997.1 UGUGUUCAAAGUCAAGGAUAU 1635-1655A-121998.1 AUAUCCUUGACUUUGAACACAUG 1633-1655 60337.1 AD- A-122027.1AUUGAUGAGAUCCGGGACUUG 1486-1506 A-122028.1 CAAGUCCCGGAUCUCAUCAAUGA1484-1506 60333.1 AD- A-122005.1 CUGUGAGAGAGAUGCUCAAUA 2247-2267A-122006.1 UAUUGAGCAUCUCUCUCACAGCU 2245-2267 60314.1 AD- A-122007.1GAGCCAAAAAGUGUCUAGUCA 1145-1165 A-122008.1 UGACUAGACACUUUUUGGCUCCU1143-1165 60320.1 AD- A-122029.1 UCCAAGAUGAGGAUUUGGGUU 2549-2569A-122030.1 AACCCAAAUCCUCAUCUUGGAGU 2547-2569 60339.1 AD- A-122013.1CCCUUGAUAGUUCACAAGAGA 2386-2406 A-122014.1 UCUCUUGUGAACUAUCAAGGGGC2384-2406 60338.1 AD- A-121987.1 CAAAGUCAAGGAUAUGGAAAA 1641-1661A-121988.1 UUUUCCAUAUCCUUGACUUUGAA 1639-1661 60307.1 AD- A-122019.1UAGUUCACAAGAGAAGUCGUU 2393-2413 A-122020.1 AACGACUUCUCUUGUGAACUAUC2391-2413 60309.1 AD- A-121999.1 GGCCCCUUGAUAGUUCACAAG 2383-2403A-122000.1 CUUGUGAACUAUCAAGGGGCCGC 2381-2403 60343.1 AD- A-121977.1UGGUGCUAGAUGGAUCAGACA 1100-1120 A-121978.1 UGUCUGAUCCAUCUAGCACCAGG1098-1120 60324.1 AD- A-121975.1 GCUAGAUGGAUCAGACAGCAU 1104-1124A-121976.1 AUGCUGUCUGAUCCAUCUAGCAC 1102-1124 60318.1 AD- A-121969.1UACCUGGUGCUAGAUGGAUCA 1096-1116 A-121970.1 UGAUCCAUCUAGCACCAGGUAGA1094-1116 60300.1 AD- A-121979.1 GGUGCUAGAUGGAUCAGACAA 1101-1121A-121980.1 UUGUCUGAUCCAUCUAGCACCAG 1099-1121 60330.1 (G19A) (G19A) AD-A-121971.1 UCUGAGUCUCUGUGGCAUGGU 1704-1724 A-121972.1ACCAUGCCACAGAGACUCAGAGA 1702-1724 60306.1 AD- A-121981.1GUGCUAGAUGGAUCAGACAGA 1102-1122 A-121982.1 UCUGUCUGAUCCAUCUAGCACCA1100-1122 60336.1 (C19A) (C19A) AD- A-121985.1 CUACCUGGUGCUAGAUGGAUA1095-1115 A-121986.1 UAUCCAUCUAGCACCAGGUAGAU 1093-1115 60301.1 (C19A)(C19A) AD- A-121983.1 ACCUGGUGCUAGAUGGAUCAA 1097-1117 A-121984.1UUGAUCCAUCUAGCACCAGGUAG 1095-1117 60342.1 (G19A) (G19A)Rodent CFB Sequences Sense Sequence Antisense Sequence(SEQ ID NOS: 103-117, (SEQ ID NOS: 118-132, Duplex respectively,Position in Antisense respectively, Position in ID Sense IDin order of appearance) NM_001142706.1 ID in order of appearance)NM_001142706.1 AD- A-122043.1 GCAAGCCAAGAUCUCAGUCAC 1888-1908 A-122044.1GUGACUGAGAUCUUGGCUUGCCA 1886-1908 60334.1 AD- A-122033.1GAUUGAGAAGGUGGCGAGUUA 1291-1311 A-122034.1 UAACUCGCCACCUUCUCAAUCAA1289-1311 60304.1 AD- A-122035.1 CACAAGAGAAGCCGCUUCAUU 2515-2535A-122036.1 AAUGAAGCGGCUUCUCUUGUGAA 2513-2535 60310.1 AD- A-122041.1UUGUGAGAGAGAUGCUACAAA 2364-2384 A-122042.1 UUUGUAGCAUCUCUCUCACAACU2362-2384 60328.1 AD- A-122039.1 UCCUUCAUGAAUGUUCCGGGA 193-213A-122040.1 UCCCGGAACAUUCAUGAAGGAGG 191-213 60322.1 AD- A-122037.1UCACAGAGAAGCUCAACCAAA 1407-1427 A-122038.1 UUUGGUUGAGCUUCUCUGUGACC1405-1427 60316.1 AD- A-122047.1 CUCAACCAAAUCAGUUAUGAA 1418-1438A-122048.1 UUCAUAACUGAUUUGGUUGAGCU 1416-1438 60346.1 AD- A-122059.1CCCUGACAGAGACCAUCGAAG 1113-1133 A-122060.1 CUUCGAUGGUCUCUGUCAGGGAG1111-1133 60335.1 AD- A-122055.1 GAGCAGAUUGCAUAAAAGGUU 261-281A-122056.1 AACCUUUUAUGCAAUCUGCUCUG 259-281 60323.1 AD- A-122045.1CUUCAUGAAUGUUCCGGGAAG 195-215 A-122046.1 CUUCCCGGAACAUUCAUGAAGGA 193-21560340.1 AD- A-122049.1 CUUCAUUCAAGUUGGUGUGAU 2529-2549 A-122050.1AUCACACCAACUUGAAUGAAGCG 2527-2549 60305.1 AD- A-122053.1GAUUGAAGAGGUCCUGUUCCA 2050-2070 A-122054.1 UGGAACAGGACCUCUUCAAUCUC2048-2070 60317.1 AD- A-122057.1 AUUUCUUUUCAAUGCUAUGAU 782-802A-122058.1 AUCAUAGCAUUGAAAAGAAAUCU 780-802 60329.1 AD- A-122061.1CCAGAGCAGAUUGCAUAAAAG 258-278 A-122062.1 CUUUUAUGCAAUCUGCUCUGGCA 256-27860341.1 AD- A-122051.1 CACAGAGAAGCUCAACCAAAU 1408-1428 A-122052.1AUUUGGUUGAGCUUCUCUGUGAC 1406-1428 60311.1

TABLE 4 Complement Factor B (CFB) modified sequences Human CFB SequencesSense Sequence Antisense Sequence (SEQ ID NOS 133-163, respectively,Antisense (SEQ ID NOS 164-194, respectively, Duplex ID Sense IDin order of appearance) ID in order of appearance) AD-60315.1 A-122021.1AfsusUfcCfuGfaAfUfUfuUfaUfgAfcUfaUfL96 A-122022.1asUfsaGfuCfaUfaAfaauUfcAfgGfa Afususc AD-60326.1 A-122009.1CfscsUfgAfuCfaAfGfCfuCfaAfgAfaUfaAfL96 A-122010.1usUfsaUfuCfuUfgAfgcuUfgAfuCfa Gfgsgsc AD-60303.1 A-122017.1GfsasAfgCfaGfgAfAfUfuCfcUfgAfaUfuUfL96 A-122018.1asAfsaUfuCfaGfgAfauuCfcUfgCfu Ufcsusu AD-60331.1 A-121995.1AfsgsCfaAfcAfuGfUfGfuUfcAfaAfgUfcAfL96 A-121996.1usGfsaCfuUfuGfaAfcacAfuGfuUfg Cfuscsa AD-60344.1 A-122015.1GfscsUfgUfgGfuGfUfCfuGfaGfuAfcUfuUfL96 A-122016.1asAfsaGfuAfcUfcAfgacAfcCfaCfa Gfcscsc AD-60345.1 A-122031.1AfsasGfuGfuCfuAfGfUfcAfaCfuUfaAfuUfL96 A-122032.1asAfsuUfaAfgUfuGfacuAfgAfcAfc Ufususu AD-60319.1 A-121991.1AfsgsCfuGfuGfaGfAfGfaGfaUfgCfuCfaAfL96 A-121992.1usUfsgAfgCfaUfcUfcucUfcAfcAfg Cfusgsc AD-60308.1 A-122003.1AfsgsCfcAfaAfaAfGfUfgUfcUfaGfuCfaAfL96 A-122004.1usUfsgAfcUfaGfaCfacuUfuUfuGfg Cfuscsc AD-60332.1 A-122011.1UfsgsUfgAfgUfgAfUfGfaGfaUfcUfcUfuUfL96 A-122012.1asAfsaGfaGfaUfcUfcauCfaCfuCfa Cfasusu AD-60313.1 A-121989.1AfsasUfuGfaGfaAfGfGfuGfgCfaAfgUfuAfL96 A-121990.1usAfsaCfuUfgCfcAfccuUfcUfcAfa Ufusasa AD-60321.1 A-122023.1CfsasAfcAfuGfuGfUfUfcAfaAfgUfcAfaGfL96 A-122024.1csUfsuGfaCfuUfuGfaacAfcAfuGfu Ufgscsu AD-60327.1 A-122025.1UfsgsUfgAfgAfgAfGfAfuGfcUfcAfaUfaUfL96 A-122026.1asUfsaUfuGfaGfcAfucuCfuCfuCfa Cfasgsc AD-60302.1 A-122001.1GfsusCfuAfgUfcAfAfCfuUfaAfuUfgAfgAfL96 A-122002.1usCfsuCfaAfuUfaAfguuGfaCfuAfg Afcsasc AD-60325.1 A-121993.1UfscsCfaAfgAfaAfGfAfcAfaUfgAfgCfaAfL96 A-121994.1usUfsgCfuCfaUfuGfucuUfuCfuUfg Gfasasg AD-60337.1 A-121997.1UfsgsUfgUfuCfaAfAfGfuCfaAfgGfaUfaUfL96 A-121998.1asUfsaUfcCfuUfgAfcuuUfgAfaCfa Cfasusg AD-60333.1 A-122027.1AfsusUfgAfuGfaGfAfUfcCfgGfgAfcUfuGfL96 A-122028.1csAfsaGfuCfcCfgGfaucUfcAfuCfa Afusgsa AD-60314.1 A-122005.1CfsusGfuGfaGfaGfAfGfaUfgCfuCfaAfuAfL96 A-122006.1usAfsuUfgAfgCfaUfcucUfcUfcAfc Afgscsu AD-60320.1 A-122007.1GfsasGfcCfaAfaAfAfGfuGfuCfuAfgUfcAfL96 A-122008.1usGfsaCfuAfgAfcAfcuuUfuUfgGfc Ufcscsu AD-60339.1 A-122029.1UfscsCfaAfgAfuGfAfGfgAfuUfuGfgGfuUfL96 A-122030.1asAfscCfcAfaAfuCfcucAfuCfuUfg Gfasgsu AD-60338.1 A-122013.1CfscsCfuUfgAfuAfGfUfuCfaCfaAfgAfgAfL96 A-122014.1usCfsuCfuUfgUfgAfacuAfuCfaAfg Gfgsgsc AD-60307.1 A-121987.1CfsasAfaGfuCfaAfGfGfaUfaUfgGfaAfaAfL96 A-121988.1usUfsuUfcCfaUfaUfccuUfgAfcUfu Ufgsasa AD-60309.1 A-122019.1UfsasGfuUfcAfcAfAfGfaGfaAfgUfcGfuUfL96 A-122020.1asAfscGfaCfuUfcUfcuuGfuGfaAfc Ufasusc AD-60343.1 A-121999.1GfsgsCfcCfcUfuGfAfUfaGfuUfcAfcAfaGfL96 A-122000.1csUfsuGfuGfaAfcUfaucAfaGfgGfg Cfcsgsc AD-60324.1 A-121977.1UfsgsGfuGfcUfaGfAfUfgGfaUfcAfgAfcAfL96 A-121978.1usGfsuCfuGfaUfcCfaucUfaGfcAfc Cfasgsg AD-60318.1 A-121975.1GfscsUfaGfaUfgGfAfUfcAfgAfcAfgCfaUfL96 A-121976.1asUfsgCfuGfuCfuGfaucCfaUfcUfa Gfcsasc AD-60300.1 A-121969.1UfsasCfcUfgGfuGfCfUfaGfaUfgGfaUfcAfL96 A-121970.1usGfsaUfcCfaUfcUfagcAfcCfaGfg Ufasgsa AD-60330.1 A-121979.1GfsgsUfgCfuAfgAfUfGfgAfuCfaGfaCfaAfL96 A-121980.1usUfsgUfcUfgAfuCfcauCfuAfgCfa Cfcsasg AD-60306.1 A-121971.1UfscsUfgAfgUfcUfCfUfgUfgGfcAfuGfgUfL96 A-121972.1asCfscAfuGfcCfaCfagaGfaCfuCfa Gfasgsa AD-60336.1 A-121981.1GfsusGfcUfaGfaUfGfGfaUfcAfgAfcAfgAfL96 A-121982.1usCfsuGfuCfuGfaUfccaUfcUfaGfc Afcscsa AD-60301.1 A-121985.1CfsusAfcCfuGfgUfGfCfuAfgAfuGfgAfuAfL96 A-121986.1usAfsuCfcAfuCfuAfgcaCfcAfgGfu Afgsasu AD-60342.1 A-121983.1AfscsCfuGfgUfgCfUfAfgAfuGfgAfuCfaAfL96 A-121984.1usUfsgAfuCfcAfuCfuagCfaCfcAfg Gfusasg Rodent CFB SequencesSense Sequence Antisense Sequence (SEQ ID NOS 195-209, respectively,Antisense (SEQ ID NOS 210-224, respectively, Duplex ID Sense IDin order of appearance) ID in order of appearance) AD-60334.1 A-122043.1GfscsAfaGfcCfaAfGfAfuCfuCfaGfuCfaCfL96 A-122044.1gsUfsgAfcUfgAfgAfucuUfgGfcUfu Gfcscsa AD-60304.1 A-122033.1GfsasUfuGfaGfaAfGfGfuGfgCfgAfgUfuAfL96 A-122034.1usAfsaCfuCfgCfcAfccuUfcUfcAfa Ufcsasa AD-60310.1 A-122035.1CfsasCfaAfgAfgAfAfGfcCfgCfuUfcAfuUfL96 A-122036.1asAfsuGfaAfgCfgGfcuuCfuCfuUfg Ufgsasa AD-60328.1 A-122041.1UfsusGfuGfaGfaGfAfGfaUfgCfuAfcAfaAfL96 A-122042.1usUfsuGfuAfgCfaUfcucUfcUfcAfc Afascsu AD-60322.1 A-122039.1UfscsCfuUfcAfuGfAfAfuGfuUfcCfgGfgAfL96 A-122040.1usCfscCfgGfaAfcAfuucAfuGfaAfg Gfasgsg AD-60316.1 A-122037.1UfscsAfcAfgAfgAfAfGfcUfcAfaCfcAfaAfL96 A-122038.1usUfsuGfgUfuGfaGfcuuCfuCfuGfu Gfascsc AD-60346.1 A-122047.1CfsusCfaAfcCfaAfAfUfcAfgUfuAfuGfaAfL96 A-122048.1usUfscAfuAfaCfuGfauuUfgGfuUfg Afgscsu AD-60335.1 A-122059.1CfscsCfuGfaCfaGfAfGfaCfcAfuCfgAfaGfL96 A-122060.1csUfsuCfgAfuGfgUfcucUfgUfcAfg Gfgsasg AD-60323.1 A-122055.1GfsasGfcAfgAfuUfGfCfaUfaAfaAfgGfuUfL96 A-122056.1asAfscCfuUfuUfaUfgcaAfuCfuGfc Ufcsusg AD-60340.1 A-122045.1CfsusUfcAfuGfaAfUfGfuUfcCfgGfgAfaGfL96 A-122046.1csUfsuCfcCfgGfaAfcauUfcAfuGfa Afgsgsa AD-60305.1 A-122049.1CfsusUfcAfuUfcAfAfGfuUfgGfuGfuGfaUfL96 A-122050.1asUfscAfcAfcCfaAfcuuGfaAfuGfa Afgscsg AD-60317.1 A-122053.1GfsasUfuGfaAfgAfGfGfuCfcUfgUfuCfcAfL96 A-122054.1usGfsgAfaCfaGfgAfccuCfuUfcAfa Ufcsusc AD-60329.1 A-122057.1AfsusUfuCfuUfuUfCfAfaUfgCfuAfuGfaUfL96 A-122058.1asUfscAfuAfgCfaUfugaAfaAfgAfa Afuscsu AD-60341.1 A-122061.1CfscsAfgAfgCfaGfAfUfuGfcAfuAfaAfaGfL96 A-122062.1csUfsuUfuAfuGfcAfaucUfgCfuCfu Gfgscsa AD-60311.1 A-122051.1CfsasCfaGfaGfaAfGfCfuCfaAfcCfaAfaUfL96 A-122052.1asUfsuUfgGfuUfgAfgcuUfcUfcUfg Ufgsasc

TABLE 5 C3 unmodified sequences Sense Sequence (SEQ ID NOS 225-265,respectively, in Position in Duplex ID Sense ID order of appearance)NM_000064.2 AD-60149.1 A-121853.1 CGUGGUCAAGGUCUUCUCUCU 3309-3329AD-60151.1 A-121885.1 ACGUGGUCAAGGUCUUCUCUA 3308-3324_C21A AD-60152.1A-121901.1 UUUGACCUCAUGGUGUUCGUG 1174-1194 AD-60153.1 A-121917.1GGAGAAUUGCUUCAUACAAAA 4611-4631 AD-60154.1 A-121933.1UGUUAAAUGGCUGAUCCUGGA 3375-3395 AD-60155.1 A-121855.1GACAGACAAGACCAUCUACAC 465-485 AD-60156.1 A-121871.1CCAGACAGACAAGACCAUCUA 462-482 AD-60157.1 A-121887.1CCAGAUCCACUUCACCAAGAA 1125-1141_C21A AD-60158.1 A-121903.1UUGACCUCAUGGUGUUCGUGA 1175-1195 AD-60159.1 A-121919.1CCCCUUCGAGGUCACAGUAAU 2523-2543 AD-60160.1 A-121935.1AUGAACAAAACUGUGGCUGUU 2878-2898 AD-60161.1 A-121857.1AGACAGACAAGACCAUCUACA 464-484 AD-60162.1 A-121873.1CCAGAUCCACUUCACCAAGAC 1125-1145 AD-60163.1 A-121889.1AGGGAUCUGUGUGGCAGACCA 2505-2521_C21A AD-60164.1 A-121905.1GACAAGACCAUCUACACCCCU 469-489 AD-60165.1 A-121921.1GCUGAGGAGAAUUGCUUCAUA 4606-4626 AD-60166.1 A-121859.1ACGUGGUCAAGGUCUUCUCUC 3308-3328 AD-60167.1 A-121875.1GGAUCUGUGUGGCAGACCCCU 2507-2527 AD-60168.1 A-121891.1ACAGACAAGACCAUCUACACA 466-482_C21A AD-60169.1 A-121907.1AUCCAGACAGACAAGACCAUU 460-476_C21U AD-60170.1 A-121923.1CUCCGUGUGGGUGGACGUCAA 1713-1733 AD-60171.1 A-121861.1UCCAGACAGACAAGACCAUCU 461-481 AD-60172.1 A-121877.1AGGGAUCUGUGUGGCAGACCC 2505-2525 AD-60173.1 A-121893.1CAAGAAAGGGAUCUGUGUGGA 2499-2515_C21A AD-60174.1 A-121909.1UGACCUCAUGGUGUUCGUGAU 1176-1192_C21U AD-60175.1 A-121925.1GCAGCUAAAAGACUUUGACUU 3789-3809 AD-60176.1 A-121863.1CAUCCAGACAGACAAGACCAU 459-479 AD-60177.1 A-121879.1ACAGACAAGACCAUCUACACC 466-486 AD-60178.1 A-121895.1AUCCAGACAGACAAGACCAUC 460-480 AD-60179.1 A-121911.1UUUGACCUCAUGGUGUUCGUU 1174-1190_G21U AD-60180.1 A-121927.1GGAUGCCAAGAACACUAUGAU 4200-4220 AD-60181.1 A-121865.1AAGAAAGGGAUCUGUGUGGCA 2500-2520 AD-60182.1 A-121881.1CAAGAAAGGGAUCUGUGUGGC 2499-2519 AD-60183.1 A-121897.1UACGUGGUCAAGGUCUUCUCU 3307-3327 AD-60184.1 A-121913.1CAGUUUCGAGGUCAUAGUGGA 756-776 AD-60185.1 A-121929.1CGUGCCGGAAGGAAUCAGAAU 2859-2879 AD-60186.1 A-121867.1GAAAGGGAUCUGUGUGGCAGA 2502-2522 AD-60187.1 A-121883.1GACAGACAAGACCAUCUACAA 465-481_C21A AD-60188.1 A-121899.1UGACCUCAUGGUGUUCGUGAC 1176-1196 AD-60189.1 A-121915.1UGUAAUAAAUUCGACCUCAAG 4138-4158 AD-60190.1 A-121931.1AACUACAUGAACCUACAGAGA 3601-3621 Antisense Seqeunce (SEQ ID NOS 266-306,respectively, in order Position in Duplex ID Antisense ID of appearance)NM_000064.2 AD-60149.1 A-121854.1 AGAGAGAAGACCUUGACCACGUA 3307-3329AD-60151.1 A-121886.1 UAGAGAAGACCUUGACCACGUAG 3306-3324_C21A AD-60152.1A-121902.1 CACGAACACCAUGAGGUCAAAGG 1172-1194 AD-60153.1 A-121918.1UUUUGUAUGAAGCAAUUCUCCUC 4609-4631 AD-60154.1 A-121934.1UCCAGGAUCAGCCAUUUAACAGC 3373-3395 AD-60155.1 A-121856.1GUGUAGAUGGUCUUGUCUGUCUG 463-485 AD-60156.1 A-121872.1UAGAUGGUCUUGUCUGUCUGGAU 460-482 AD-60157.1 A-121888.1UUCUUGGUGAAGUGGAUCUGGUA 1123-1141_C21A AD-60158.1 A-121904.1UCACGAACACCAUGAGGUCAAAG 1173-1195 AD-60159.1 A-121920.1AUUACUGUGACCUCGAAGGGGUC 2521-2543 AD-60160.1 A-121936.1AACAGCCACAGUUUUGUUCAUUC 2876-2898 AD-60161.1 A-121858.1UGUAGAUGGUCUUGUCUGUCUGG 462-484 AD-60162.1 A-121874.1GUCUUGGUGAAGUGGAUCUGGUA 1123-1145 AD-60163.1 A-121890.1UGGUCUGCCACACAGAUCCCUUU 2503-2521_C21A AD-60164.1 A-121906.1AGGGGUGUAGAUGGUCUUGUCUG 467-489 AD-60165.1 A-121922.1UAUGAAGCAAUUCUCCUCAGCAC 4604-4626 AD-60166.1 A-121860.1GAGAGAAGACCUUGACCACGUAG 3306-3328 AD-60167.1 A-121876.1AGGGGUCUGCCACACAGAUCCCU 2505-2527 AD-60168.1 A-121892.1UGUGUAGAUGGUCUUGUCUGUCU 464-482_C21A AD-60169.1 A-121908.1AAUGGUCUUGUCUGUCUGGAUGA 458-476_C21U AD-60170.1 A-121924.1UUGACGUCCACCCACACGGAGUC 1711-1733 AD-60171.1 A-121862.1AGAUGGUCUUGUCUGUCUGGAUG 459-481 AD-60172.1 A-121878.1GGGUCUGCCACACAGAUCCCUUU 2503-2525 AD-60173.1 A-121894.1UCCACACAGAUCCCUUUCUUGUC 2497-2515_C21A AD-60174.1 A-121910.1AUCACGAACACCAUGAGGUCAAA 1174-1192_C21U AD-60175.1 A-121926.1AAGUCAAAGUCUUUUAGCUGCAG 3787-3809 AD-60176.1 A-121864.1AUGGUCUUGUCUGUCUGGAUGAA 457-479 AD-60177.1 A-121880.1GGUGUAGAUGGUCUUGUCUGUCU 464-486 AD-60178.1 A-121896.1GAUGGUCUUGUCUGUCUGGAUGA 458-480 AD-60179.1 A-121912.1AACGAACACCAUGAGGUCAAAGG 1172-1190_G21U AD-60180.1 A-121928.1AUCAUAGUGUUCUUGGCAUCCUG 4198-4220 AD-60181.1 A-121866.1UGCCACACAGAUCCCUUUCUUGU 2498-2520 AD-60182.1 A-121882.1GCCACACAGAUCCCUUUCUUGUC 2497-2519 AD-60183.1 A-121898.1AGAGAAGACCUUGACCACGUAGG 3305-3327 AD-60184.1 A-121914.1UCCACUAUGACCUCGAAACUGGG 754-776 AD-60185.1 A-121930.1AUUCUGAUUCCUUCCGGCACGAC 2857-2879 AD-60186.1 A-121868.1UCUGCCACACAGAUCCCUUUCUU 2500-2522 AD-60187.1 A-121884.1UUGUAGAUGGUCUUGUCUGUCUG 463-481_C21A AD-60188.1 A-121900.1GUCACGAACACCAUGAGGUCAAA 1174-1196 AD-60189.1 A-121916.1CUUGAGGUCGAAUUUAUUACAGG 4136-4158 AD-60190.1 A-121932.1UCUCUGUAGGUUCAUGUAGUUGG 3599-3621

TABLE 6 C3 modified sequences Antisense Sequence Sense Sequence(SEQ ID NOS 348-388, (SEQ ID NOS 308-347, respectively, Antisenserespectively, Duplex ID Sense ID in order of appearance) IDin order of appearance) AD-60149.1 A-121853.1CfsgsUfgGfuCfaAfGfGfuCfuUfcUfcUfcUfL96 A-121854.1asGfsaGfaGfaAfgAfccuUfgAfcCfa Cfgsusa AD-60151.1 A-121885.1AfscsGfuGfgUfcAfAfGfgUfcUfuCfuCfuAfL96 A-121886.1usAfsgAfgAfaGfaCfcuuGfaCfcAfc Gfusasg AD-60152.1 A-121901.1UfsusUfgAfcCfuCfAfUfgGfuGfuUfcGfuGfL96 A-121902.1csAfscGfaAfcAfcCfaugAfgGfuCfa Afasgsg AD-60153.1 A-121917.1GfsgsAfgAfaUfuGfCfUfuCfaUfaCfaAfaAfL96 A-121918.1usUfsuUfgUfaUfgAfagcAfaUfuCfu Cfcsusc AD-60154.1 A-121933.1UfsgsUfuAfaAfuGfGfCfuGfaUfcCfuGfgAfL96 A-121934.1usCfscAfgGfaUfcAfgccAfuUfuAfa Cfasgsc AD-60155.1 A-121855.1GfsasCfaGfaCfaAfGfAfcCfaUfcUfaCfaCfL96 A-121856.1gsUfsgUfaGfaUfgGfucuUfgUfcUfg Ufcsusg AD-60156.1 A-121871.1CfscsAfgAfcAfgAfCfAfaGfaCfcAfuCfuAfL96 A-121872.1usAfsgAfuGfgUfcUfuguCfuGfuCfu Gfgsasu AD-60157.1 A-121887.1CfscsAfgAfuCfcAfCfUfuCfaCfcAfaGfaAfL96 A-121888.1usUfscUfuGfgUfgAfaguGfgAfuCfu Gfgsusa AD-60158.1 A-121903.1UfsusGfaCfcUfcAfUfGfgUfgUfuCfgUfgAfL96 A-121904.1usCfsaCfgAfaCfaCfcauGfaGfgUfc Afasasg AD-60159.1 A-121919.1CfscsCfcUfuCfgAfGfGfuCfaCfaGfuAfaUfL96 A-121920.1asUfsuAfcUfgUfgAfccuCfgAfaGfg Gfgsusc AD-60160.1 A-121935.1AfsusGfaAfcAfaAfAfCfuGfuGfgCfuGfuUfL96 A-121936.1asAfscAfgCfcAfcAfguuUfuGfuUfc Afususc AD-60161.1 A-121857.1AfsgsAfcAfgAfcAfAfGfaCfcAfuCfuAfcAfL96 A-121858.1usGfsuAfgAfuGfgUfcuuGfuCfuGfu Cfusgsg AD-60162.1 A-121873.1CfscsAfgAfuCfcAfCfUfuCfaCfcAfaGfaCfL96 A-121874.1gsUfscUfuGfgUfgAfaguGfgAfuCfu Gfgsusa AD-60163.1 A-121889.1AfsgsGfgAfuCfuGfUfGfuGfgCfaGfaCfcAfL96 A-121890.1usGfsgUfcUfgCfcAfcacAfgAfuCfc Cfususu AD-60164.1 A-121905.1GfsasCfaAfgAfcCfAfUfcUfaCfaCfcCfcUfL96 A-121906.1asGfsgGfgUfgUfaGfaugGfuCfuUfg Ufcsusg AD-60165.1 A-121921.1GfscsUfgAfgGfaGfAfAfuUfgCfuUfcAfuAfL96 A-121922.1usAfsuGfaAfgCfaAfuucUfcCfuCfa Gfcsasc AD-60166.1 A-121859.1AfscsGfuGfgUfcAfAfGfgUfcUfuCfuCfuCfL96 A-121860.1gsAfsgAfgAfaGfaCfcuuGfaCfcAfc Gfusasg AD-60167.1 A-121875.1GfsgsAfuCfuGfuGfUfGfgCfaGfaCfcCfcUfL96 A-121876.1asGfsgGfgUfcUfgCfcacAfcAfgAfu Cfcscsu AD-60168.1 A-121891.1AfscsAfgAfcAfaGfAfCfcAfuCfuAfcAfcAfL96 A-121892.1usGfsuGfuAfgAfuGfgucUfuGfuCfu Gfuscsu AD-60169.1 A-121907.1AfsusCfcAfgAfcAfGfAfcAfaGfaCfcAfuUfL96 A-121908.1asAfsuGfgUfcUfuGfucuGfuCfuGfg Afusgsa AD-60170.1 A-121923.1CfsusCfcGfuGfuGfGfGfuGfgAfcGfuCfaAfL96 A-121924.1usUfsgAfcGfuCfcAfcccAfcAfcGfg Afgsusc AD-60171.1 A-121861.1UfscsCfaGfaCfaGfAfCfaAfgAfcCfaUfcUfL96 A-121862.1asGfsaUfgGfuCfuUfgucUfgUfcUfg Gfasusg AD-60172.1 A-121877.1AfsgsGfgAfuCfuGfUfGfuGfgCfaGfaCfcCfL96 A-121878.1gsGfsgUfcUfgCfcAfcacAfgAfuCfc Cfususu AD-60173.1 A-121893.1CfsasAfgAfaAfgGfGfAfuCfuGfuGfuGfgAfL96 A-121894.1usCfscAfcAfcAfgAfuccCfuUfuCfu Ufgsusc AD-60174.1 A-121909.1UfsgsAfcCfuCfaUfGfGfuGfuUfcGfuGfaUfL96 A-121910.1asUfscAfcGfaAfcAfccaUfgAfgGfu Cfasasa AD-60175.1 A-121925.1GfscsAfgCfuAfaAfAfGfaCfuUfuGfaCfuUfL96 A-121926.1asAfsgUfcAfaAfgUfcuuUfuAfgCfu Gfcsasg AD-60176.1 A-121863.1CfsasUfcCfaGfaCfAfGfaCfaAfgAfcCfaUfL96 A-121864.1asUfsgGfuCfuUfgUfcugUfcUfgGfa Ufgsasa AD-60177.1 A-121879.1AfscsAfgAfcAfaGfAfCfcAfuCfuAfcAfcCfL96 A-121880.1gsGfsuGfuAfgAfuGfgucUfuGfuCfu Gfuscsu AD-60178.1 A-121895.1AfsusCfcAfgAfcAfGfAfcAfaGfaCfcAfuCfL96 A-121896.1gsAfsuGfgUfcUfuGfucuGfuCfuGfg Afusgsa AD-60179.1 A-121911.1UfsusUfgAfcCfuCfAfUfgGfuGfuUfcGfuUfL96 A-121912.1asAfscGfaAfcAfcCfaugAfgGfuCfa Afasgsg AD-60180.1 A-121927.1GfsgsAfuGfcCfaAfGfAfaCfaCfuAfuGfaUfL96 A-121928.1asUfscAfuAfgUfgUfucuUfgGfcAfu Cfcsusg AD-60181.1 A-121865.1AfsasGfaAfaGfgGfAfUfcUfgUfgUfgGfcAfL96 A-121866.1usGfscCfaCfaCfaGfaucCfcUfuUfc Ufusgsu AD-60182.1 A-121881.1CfsasAfgAfaAfgGfGfAfuCfuGfuGfuGfgCfL96 A-121882.1gsCfscAfcAfcAfgAfuccCfuUfuCfu Ufgsusc AD-60183.1 A-121897.1UfsasCfgUfgGfuCfAfAfgGfuCfuUfcUfcUfL96 A-121898.1asGfsaGfaAfgAfcCfuugAfcCfaCfg Ufasgsg AD-60184.1 A-121913.1CfsasGfuUfuCfgAfGfGfuCfaUfaGfuGfgAfL96 A-121914.1usCfscAfcUfaUfgAfccuCfgAfaAfc Ufgsgsg AD-60185.1 A-121929.1CfsgsUfgCfcGfgAfAfGfgAfaUfcAfgAfaUfL96 A-121930.1asUfsuCfuGfaUfuCfcuuCfcGfgCfa Cfgsasc AD-60186.1 A-121867.1GfsasAfaGfgGfaUfCfUfgUfgUfgGfcAfgAfL96 A-121868.1usCfsuGfcCfaCfaCfagaUfcCfcUfu Ufcsusu AD-60187.1 A-121883.1GfsasCfaGfaCfaAfGfAfcCfaUfcUfaCfaAfL96 A-121884.1usUfsgUfaGfaUfgGfucuUfgUfcUfg Ufcsusg AD-60188.1 A-121899.1UfsgsAfcCfuCfaUfGfGfuGfuUfcGfuGfaCfL96 A-121900.1gsUfscAfcGfaAfcAfccaUfgAfgGfu Cfasasa AD-60189.1 A-121915.1UfsgsUfaAfuAfaAfUfUfcGfaCfcUfcAfaGfL96 A-121916.1csUfsuGfaGfgUfcGfaauUfuAfuUfa Cfasgsg AD-60190.1 A-121931.1AfsasCfuAfcAfuGfAfAfcCfuAfcAfgAfgAfL96 A-121932.1usCfsuCfuGfuAfgGfuucAfuGfuAfg Ufusgsg

TABLE 7 C9 unmodified sequences Sense Seqeunce Antisense Seqeunce(SEQ ID NOS 389-411, (SEQ ID NOS 412-434, respectively, in Position inAntisense respectively, Position in Duplex ID Sense IDorder of appearance) NM_001737.3 ID in order of appearance) NM_001737.3AD-59663.1 A-121046.1 UUUUGACAAUGAGUUCUACAA 606-626 A-121047.1UUGUAGAACUCAUUGUCAAAAGG 604-626 AD-59664.1 A-121062.1AUCAAUGAAUUUAGUGUAAGA 1597-1617 A-121063.1 UCUUACACUAAAUUCAUUGAUAU1595-1617 AD-59665.1 A-121078.1 AGACAAAUGUUUCGUUCAAGA 268-288 A-121079.1UCUUGAACGAAACAUUUGUCUGA 266-288 AD-59668.1 A-121048.1CUUUUGACAAUGAGUUCUACA 605-625 A-121049.1 UGUAGAACUCAUUGUCAAAAGGU 603-625AD-59669.1 A-121064.1 AACUUGGAAAGAGCCAUUGAAx 1570-1590 A-121065.1UUCAAUGGCUCUUUCCAAGUUUU 1568-1590 AD-59670.1 A-121080.1UACCUGAGAAGCUGAUUAACAx 2589-2609 A-121081.1 UGUUAAUCAGCUUCUCAGGUAGG2587-2609 AD-59673.1 A-121050.1 ACCUUUUGACAAUGAGUUCUA 603-623 A-121051.1UAGAACUCAUUGUCAAAAGGUGU 601-623 AD-59674.1 A-121066.1GACUGCGGAAAUGACUUUCAA 391-411 A-121067.1 UUGAAAGUCAUUUCCGCAGUCAU 389-411AD-59675.1 A-121082.1 GCCCAUUCAAAUUUGAGGGAA 1682-1702 A-121083.1UUCCCUCAAAUUUGAAUGGGCAG 1680-1702 AD-59678.1 A-121052.1UUUUGGAUAAAGCUUCCAUGA 1175-1195 A-121053.1 UCAUGGAAGCUUUAUCCAAAACA1173-1195 AD-59679.1 A-121068.1 AACCAAAGGCGAGAAAAAUUU 708-728 A-121069.1AAAUUUUUCUCGCCUUUGGUUUC 706-728 AD-59680.1 A-121084.1CUUUGCCAACUACCUAUGAAA 1067-1087 A-121085.1 UUUCAUAGGUAGUUGGCAAAGCU1065-1087 AD-59683.1 A-121054.1 CACCUUUUGACAAUGAGUUCU 602-622 A-121055.1AGAACUCAUUGUCAAAAGGUGUG 600-622 AD-59684.1 A-121070.1GAGAAGACAUCAAAUUUUAAU 781-801 A-121071.1 AUUAAAAUUUGAUGUCUUCUCUU 779-801AD-59685.1 A-121086.1 GACAAUGAGUUCUACAAUGGA 610-630 A-121087.1UCCAUUGUAGAACUCAUUGUCAA 608-630 AD-59688.1 A-121056.1UUUGGAUAAAGCUUCCAUGAA 1176-1196 A-121057.1 UUCAUGGAAGCUUUAUCCAAAAC1174-1196 AD-59689.1 A-121072.1 AUCUAUGAAACCAAAGGCGAG 700-720 A-121073.1CUCGCCUUUGGUUUCAUAGAUCA 698-720 AD-59690.1 A-121088.1AUAUCAAUGAAUUUAGUGUAA 1595-1615 A-121089.1 UUACACUAAAUUCAUUGAUAUAG1593-1615 AD-59692.1 A-121058.1 CACACCUUUUGACAAUGAGUU 600-620 A-121059.1AACUCAUUGUCAAAAGGUGUGCU 598-620 AD-59693.1 A-121074.1UAGGGUCUGAGACCUUUUGAA 2648-2668 A-121075.1 UUCAAAAGGUCUCAGACCCUAAG2646-2668 AD-59694.1 A-121090.1 CAAAACUUGGAAAGAGCCAUU 1567-1587A-121091.1 AAUGGCUCUUUCCAAGUUUUGUU 1565-1587 AD-59696.1 A-121060.1GCACACCUUUUGACAAUGAGUx 599-619 A-121061.1 ACUCAUUGUCAAAAGGUGUGCUU597-619 AD-59697.1 A-121076.1 UGAAACCAAAGGCGAGAAAAA 705-725 A-121077.1UUUUUCUCGCCUUUGGUUUCAUA 703-725

TABLE 8 C9 modified sequences Sense Sequence Antisense Sequence(SEQ ID NOS 435-457, respectively, Antisense(SEQ ID NOS 458-480, respectively, Duplex ID Sense IDin order of appearance) ID in order of appearance) AD-59663.1 A-121046.1UfsusUfuGfaCfaAfUfGfaGfuUfcUfaCfaAfL96 A-121047.1usUfsgUfaGfaAfcUfcauUfgUfcAfaAfasgsg AD-59664.1 A-121062.1AfsusCfaAfuGfaAfUfUfuAfgUfgUfaAfgAfL96 A-121063.1usCfsuUfaCfaCfuAfaauUfcAfuUfgAfusasu AD-59665.1 A-121078.1AfsgsAfcAfaAfuGfUfUfuCfgUfuCfaAfgAfL96 A-121079.1usCfsuUfgAfaCfgAfaacAfuUfuGfuCfusgsa AD-59668.1 A-121048.1CfsusUfuUfgAfcAfAfUfgAfgUfuCfuAfcAfL96 A-121049.1usGfsuAfgAfaCfuCfauuGfuCfaAfaAfgsgsu AD-59669.1 A-121064.1AfsasCfuUfgGfaAfAfGfaGfcCfaUfuGfaAfL96 A-121065.1usUfscAfaUfgGfcUfcuuUfcCfaAfgUfususu AD-59670.1 A-121080.1UfsasCfcUfgAfgAfAfGfcUfgAfuUfaAfcAfL96 A-121081.1usGfsuUfaAfuCfaGfcuuCfuCfaGfgUfasgsg AD-59673.1 A-121050.1AfscsCfuUfuUfgAfCfAfaUfgAfgUfuCfuAfL96 A-121051.1usAfsgAfaCfuCfaUfuguCfaAfaAfgGfusgsu AD-59674.1 A-121066.1GfsasCfuGfcGfgAfAfAfuGfaCfuUfuCfaAfL96 A-121067.1usUfsgAfaAfgUfcAfuuuCfcGfcAfgUfcsasu AD-59675.1 A-121082.1GfscsCfcAfuUfcAfAfAfuUfuGfaGfgGfaAfL96 A-121083.1usUfscCfcUfcAfaAfuuuGfaAfuGfgGfcsasg AD-59678.1 A-121052.1UfsusUfuGfgAfuAfAfAfgCfuUfcCfaUfgAfL96 A-121053.1usCfsaUfgGfaAfgCfuuuAfuCfcAfaAfascsa AD-59679.1 A-121068.1AfsasCfcAfaAfgGfCfGfaGfaAfaAfaUfuUfL96 A-121069.1asAfsaUfuUfuUfcUfcgcCfuUfuGfgUfususc AD-59680.1 A-121084.1CfsusUfuGfcCfaAfCfUfaCfcUfaUfgAfaAfL96 A-121085.1usUfsuCfaUfaGfgUfaguUfgGfcAfaAfgscsu AD-59683.1 A-121054.1CfsasCfcUfuUfuGfAfCfaAfuGfaGfuUfcUfL96 A-121055.1asGfsaAfcUfcAfuUfgucAfaAfaGfgUfgsusg AD-59684.1 A-121070.1GfsasGfaAfgAfcAfUfCfaAfaUfuUfuAfaUfL96 A-121071.1asUfsuAfaAfaUfuUfgauGfuCfuUfcUfcsusu AD-59685.1 A-121086.1GfsasCfaAfuGfaGfUfUfcUfaCfaAfuGfgAfL96 A-121087.1usCfscAfuUfgUfaGfaacUfcAfuUfgUfcsasa AD-59688.1 A-121056.1UfsusUfgGfaUfaAfAfGfcUfuCfcAfuGfaAfL96 A-121057.1usUfscAfuGfgAfaGfcuuUfaUfcCfaAfasasc AD-59689.1 A-121072.1AfsusCfuAfuGfaAfAfCfcAfaAfgGfcGfaGfL96 A-121073.1csUfscGfcCfuUfuGfguuUfcAfuAfgAfuscsa AD-59690.1 A-121088.1AfsusAfuCfaAfuGfAfAfuUfuAfgUfgUfaAfL96 A-121089.1usUfsaCfaCfuAfaAfuucAfuUfgAfuAfusasg AD-59692.1 A-121058.1CfsasCfaCfcUfuUfUfGfaCfaAfuGfaGfuUfL96 A-121059.1asAfscUfcAfuUfgUfcaaAfaGfgUfgUfgscsu AD-59693.1 A-121074.1UfsasGfgGfuCfuGfAfGfaCfcUfuUfuGfaAfL96 A-121075.1usUfscAfaAfaGfgUfcucAfgAfcCfcUfasasg AD-59694.1 A-121090.1CfsasAfaAfcUfuGfGfAfaAfgAfgCfcAfuUfL96 A-121091.1asAfsuGfgCfuCfuUfuccAfaGfuUfuUfgsusu AD-59696.1 A-121060.1GfscsAfcAfcCfuUfUfUfgAfcAfaUfgAfgUfL96 A-121061.1asCfsuCfaUfuGfuCfaaaAfgGfuGfuGfcsusu AD-59697.1 A-121076.1UfsgsAfaAfcCfaAfAfGfgCfgAfgAfaAfaAfL96 A-121077.1usUfsuUfuCfuCfgCfcuuUfgGfuUfuCfasusa

TABLE 9 CFB single dose screen in Hep3B Cells 10 nM 0.1 nM 10 nM SD 0.1nM SD AD-60315.1 22.82 17.15 20.03 9.73 AD-60326.1 9.33 17.49 0.29 4.75AD-60303.1 8.45 28.08 4.67 10.75 AD-60331.1 14.47 29.99 4.36 4.99AD-60344.1 17.61 30.59 6.96 1.70 AD-60345.1 8.98 33.88 0.65 7.11AD-60319.1 14.36 33.98 1.17 12.16 AD-60308.1 12.64 34.07 0.19 11.41AD-60332.1 20.19 35.92 3.53 3.23 AD-60313.1 23.94 38.26 19.92 13.16AD-60321.1 13.32 46.50 4.83 1.00 AD-60327.1 18.44 50.40 6.45 5.21AD-60302.1 13.82 53.31 4.21 12.46 AD-60325.1 11.73 54.59 0.27 15.34AD-60337.1 16.17 56.04 3.64 33.50 AD-60333.1 17.72 65.14 2.22 8.79AD-60314.1 27.79 67.44 2.02 9.10 AD-60320.1 18.12 85.78 5.39 33.24AD-60339.1 20.86 88.73 9.59 10.47 AD-60338.1 18.14 91.03 4.11 10.07AD-60307.1 21.76 91.13 3.49 43.21 AD-60309.1 20.64 95.13 0.34 53.77AD-60343.1 61.82 112.57 5.56 17.11 AD-60324.1 24.20 81.08 3.41 18.95AD-60318.1 43.11 99.07 13.83 17.69 AD-60300.1 35.21 111.33 5.35 12.86AD-60330.1 58.80 111.85 8.86 32.76 AD-60306.1 85.87 113.97 12.01 33.11AD-60336.1 35.90 119.80 3.75 4.92 AD-60301.1 28.95 121.90 7.73 23.23AD-60342.1 49.16 123.56 17.53 14.88 AD-60334.1 26.12 55.28 22.52 7.86AD-60304.1 20.62 74.38 4.43 16.50 AD-60310.1 18.93 77.08 0.87 35.20AD-60328.1 63.55 86.20 1.91 4.07 AD-60322.1 81.67 86.30 21.22 25.58AD-60316.1 105.01 93.22 8.55 14.39 AD-60346.1 109.11 99.09 2.07 25.51AD-60335.1 42.63 101.00 5.91 54.15 AD-60323.1 81.31 103.20 4.03 3.86AD-60340.1 50.41 109.25 20.73 1.67 AD-60305.1 30.06 114.59 5.00 17.97AD-60317.1 102.87 126.87 1.95 30.25 AD-60329.1 106.30 131.90 0.20 53.49AD-60341.1 112.98 137.99 3.94 31.92 AD-60311.1 162.39 140.07 10.04 63.65

TABLE 10 CFB single dose screen in Primary Mouse Hepatocytes Avg 10 nMAvg 0.1 nM 10 nM SD 0.1 nM SD AD-60302.1 112.73 109.72 15.29 1.75AD-60303.1 119.44 102.70 0.15 23.82 AD-60307.1 67.92 99.67 2.91 6.47AD-60308.1 116.89 111.68 12.15 4.51 AD-60309.1 100.72 112.85 10.72 4.84AD-60313.1 50.21 102.05 10.08 4.13 AD-60314.1 74.12 113.15 4.99 12.59AD-60315.1 101.22 104.79 6.07 29.27 AD-60319.1 18.56 81.28 4.22 6.27AD-60320.1 103.08 123.28 8.71 18.51 AD-60321.1 45.03 104.98 3.91 25.35AD-60325.1 121.99 127.67 4.63 24.72 AD-60326.1 55.24 102.10 4.66 13.35AD-60327.1 79.42 108.21 4.77 21.99 AD-60331.1 4.51 52.03 0.35 8.06AD-60332.1 115.05 120.93 6.06 4.00 AD-60333.1 102.19 113.88 0.38 31.81AD-60337.1 3.93 31.08 1.12 0.49 AD-60338.1 120.85 115.74 9.02 8.93AD-60339.1 16.97 75.02 0.27 10.17 AD-60343.1 126.10 131.79 24.11 14.66AD-60344.1 8.06 35.14 0.31 11.86 AD-60345.1 132.64 133.75 7.96 27.82AD-60300.1 27.05 81.40 8.63 8.86 AD-60301.1 10.24 72.49 0.46 5.41AD-60306.1 97.07 114.32 4.87 18.27 AD-60318.1 37.73 98.00 3.09 7.56AD-60324.1 42.83 99.93 1.21 12.09 AD-60330.1 70.05 116.47 1.46 15.23AD-60336.1 31.97 95.19 13.63 1.75 AD-60342.1 38.22 108.31 4.90 6.76AD-60304.1 7.88 18.03 3.57 18.03 AD-60305.1 13.09 64.61 2.19 11.26AD-60310.1 1.36 21.17 0.24 1.27 AD-60311.1 2.11 28.70 0.22 4.79AD-60316.1 2.23 28.29 1.11 4.66 AD-60317.1 60.25 84.11 5.23 5.66AD-60322.1 70.53 115.47 1.47 11.72 AD-60323.1 108.71 117.31 17.38 7.90AD-60328.1 4.04 38.52 0.21 10.03 AD-60329.1 6.73 36.47 0.21 8.72AD-60334.1 49.74 99.41 2.74 8.64 AD-60335.1 34.99 99.57 3.64 1.59AD-60340.1 99.13 106.94 5.71 9.81 AD-60341.1 92.74 112.17 0.34 8.10AD-60346.1 5.65 53.30 0.52 5.28

TABLE 11 CFB Dose response screen in Hep 3B cells Duplex ID Hep3BIC50(nM) AD-60303.1 0.119 AD-60326.1 0.062 AD-60319.1 0.351 AD-60331.10.225 AD-60337.1 0.418 AD-60344.1 0.347 AD-60304.1 >10 AD-60324.1 7.039

TABLE 12 CFB Dose response screen in Primary Mouse Hepatocytes Duplex IDPrimaryMouse IC50(nM) AD-60303.1 Not achieved AD-60326.1 4.063AD-60319.1 0.162 AD-60331.1 0.031 AD-60337.1 0.014 AD-60344.1 0.003AD-60304.1 0.028 AD-60324.1 0.854

TABLE 13 C9 Single dose screen in Primary Mouse Hepatocytes Avg AvgDuplex ID 10 nM 0.1 nM SD 10 nM SD 0.1 nM AD-59663.1 5.92 27.33 2.1316.40 AD-59664.1 83.71 76.56 42.80 21.75 AD-59665.1 91.76 85.56 20.6226.31 AD-59668.1 30.66 49.06 4.23 13.47 AD-59669.1 95.36 64.74 18.6919.30 AD-59670.1 96.91 103.65 26.38 7.23 AD-59673.1 22.34 31.20 7.3420.44 AD-59674.1 12.16 45.36 5.13 14.79 AD-59675.1 93.18 109.59 3.778.45 AD-59678.1 47.33 47.23 14.22 6.86 AD-59679.1 98.53 30.06 12.8832.30 AD-59680.1 33.75 86.68 1.20 28.07 AD-59683.1 25.81 44.31 9.7823.12 AD-59684.1 58.89 96.75 16.45 21.05 AD-59685.1 68.90 115.36 8.176.36 AD-59688.1 32.69 41.63 6.49 21.72 AD-59689.1 86.86 102.46 24.470.38 AD-59690.1 101.98 131.95 4.87 0.16 AD-59692.1 33.98 36.81 9.73 3.38AD-59693.1 84.70 75.60 35.91 16.09 AD-59694.1 108.88 132.73 2.53 45.43AD-59696.1 32.87 45.82 9.72 15.79 AD-59697.1 110.00 120.20 1.21 3.98AD-1955 109.44 92.04 24.08 32.14 AD-1955 105.93 104.33 4.54 6.01 AD-195587.62 93.01 6.11 3.30 AD-1955 90.95 117.91 3.90 29.31 AD-1955 91.0493.49 6.80 8.35 AD-1955 106.63 107.78 1.44 9.89 AD-1955 95.33 82.10 9.452.92 AD-1955 123.15 121.27 44.13 11.42

TABLE 14 C3 Single dose screen in Primary Mouse Hepatocytes Avg DuplexID 10 nM Avg 0.1 nM 10 nM SD 0.1 nM SD AD-60149.1 0.08 33.89 0.04 44.73AD-60151.1 0.11 81.49 0.14 7.88 AD-60152.1 1.72 92.02 0.89 9.34AD-60153.1 93.57 97.06 17.16 4.16 AD-60154.1 97.73 122.73 0.66 28.17AD-60155.1 12.94 91.38 17.39 9.28 AD-60156.1 8.02 41.58 9.16 56.27AD-60157.1 23.61 98.22 33.22 8.77 AD-60158.1 0.75 77.42 0.76 8.61AD-60159.1 100.47 93.53 11.61 7.44 AD-60160.1 89.34 92.97 18.42 9.21AD-60161.1 2.33 82.37 0.32 21.06 AD-60162.1 60.59 46.83 1.37 65.96AD-60163.1 104.09 53.32 5.42 75.38 AD-60164.1 61.13 40.41 5.57 57.13AD-60165.1 61.93 86.61 4.44 11.53 AD-60166.1 2.27 96.48 0.70 17.52AD-60167.1 87.51 84.41 3.70 9.19 AD-60168.1 35.16 98.47 0.28 20.95AD-60169.1 0.42 51.78 0.13 18.79 AD-60170.1 125.00 99.12 1.46 12.72AD-60171.1 0.44 59.53 0.01 1.82 AD-60172.1 89.05 102.11 4.20 10.62AD-60173.1 81.29 95.39 16.08 3.86 AD-60174.1 0.06 25.26 0.02 31.64AD-60175.1 0.89 80.59 0.23 6.61 AD-60176.1 0.88 52.71 0.02 6.12AD-60177.1 63.14 85.00 16.41 9.25 AD-60178.1 42.97 64.33 4.75 14.00AD-60179.1 0.12 54.36 0.01 6.05 AD-60180.1 94.57 98.11 13.68 5.65AD-60181.1 69.28 85.66 6.99 31.48 AD-60182.1 84.22 79.05 2.63 8.99AD-60183.1 0.08 44.17 0.05 7.27 AD-60184.1 80.50 81.13 9.59 14.73AD-60185.1 92.21 99.75 12.00 2.32 AD-60186.1 60.60 93.85 18.81 29.73AD-60187.1 2.33 71.77 0.20 1.49 AD-60188.1 0.33 78.13 0.37 14.56AD-60189.1 57.75 91.38 43.16 14.16 AD-60190.1 29.40 94.84 41.57 7.55AD-1955 103.85 90.86 8.96 3.45 AD-1955 71.27 115.36 36.17 13.40 AD-195599.16 95.85 5.16 8.09 AD-1955 112.29 104.37 3.65 12.88 AD-1955 108.4497.01 1.40 0.36 AD-1955 118.26 109.90 2.10 12.76 AD-1955 98.09 98.7211.81 1.81

TABLE 15 C3 Single dose screen in Hep 3B cells Avg 10 nM 0.1 nM DuplexID Avg 10 nM 0.1 nM SD SD AD-60149.1 7.49 55.90 7.75 4.41 AD-60151.124.05 101.65 14.22 8.27 AD-60152.1 16.58 112.51 10.66 19.82 AD-60153.120.13 22.40 22.87 3.76 AD-60154.1 24.21 112.90 8.93 25.58 AD-60155.120.48 68.97 2.10 1.73 AD-60156.1 18.22 66.39 0.80 1.67 AD-60157.1 29.07125.72 5.80 8.08 AD-60158.1 81.03 105.18 14.03 14.20 AD-60159.1 27.5892.91 4.77 2.22 AD-60160.1 11.49 60.48 4.68 11.60 AD-60161.1 27.49 80.5710.88 16.13 AD-60162.1 49.58 89.22 3.76 6.06 AD-60163.1 91.18 99.19 5.1421.40 AD-60164.1 33.93 85.93 4.07 1.00 AD-60165.1 5.54 13.05 0.43 2.69AD-60166.1 35.21 81.66 21.31 14.48 AD-60167.1 106.64 115.02 8.09 39.17AD-60168.1 26.91 92.99 2.50 5.86 AD-60169.1 10.66 49.63 6.66 17.36AD-60170.1 52.73 104.43 2.71 22.03 AD-60171.1 23.77 60.35 7.94 7.27AD-60172.1 143.57 99.22 8.09 11.58 AD-60173.1 100.25 108.80 12.25 44.49AD-60174.1 16.68 92.68 0.45 45.25 AD-60175.1 24.94 42.14 4.74 7.68AD-60176.1 17.30 66.19 8.83 13.81 AD-60177.1 50.71 116.18 20.19 1.49AD-60178.1 22.65 90.84 5.82 15.23 AD-60179.1 15.21 85.30 3.55 23.07AD-60180.1 45.91 93.35 16.19 28.54 AD-60181.1 63.50 109.82 10.07 14.56AD-60182.1 110.82 121.62 1.09 6.78 AD-60183.1 13.82 69.24 8.64 3.35AD-60184.1 26.47 97.94 9.64 9.88 AD-60185.1 41.42 103.45 7.77 2.47AD-60186.1 72.24 88.39 6.37 51.31 AD-60187.1 9.49 51.15 3.28 11.65AD-60188.1 55.44 95.66 7.05 30.36 AD-60189.1 52.59 89.41 4.25 20.79AD-60190.1 16.67 95.38 1.22 11.83

TABLE 16 C3 Dose response screen in primary mouse hepatocytes PMH DuplexID IC50(nM) AD-60149.1 0.03 AD-60152.1 1.03 AD-60156.1 0.19 AD-60165.11.96 AD-60169.1 0.04 AD-60171.1 0.04 AD-60174.1 0.01 AD-60175.1 0.54AD-60176.1 0.05 AD-60179.1 0.03 AD-60183.1 0.03 AD-60187.1 0.24

TABLE 17 C3 Dose response screen in Hep3B cells Hep3B Duplex ID IC50(nM)AD-60149.1 0.88 AD-60152.1 2.87 AD-60156.1 2.06 AD-60165.1 0.08AD-60169.1 0.41 AD-60171.1 5.51 AD-60174.1 2.60 AD-60175.1 0.48AD-60176.1 2.29 AD-60179.1 1.70 AD-60183.1 0.94 AD-60187.1 1.65

Example 3. In Vivo Screening

A subset of three CFB GalNAC conjugated iRNAs was selected for furtherin vivo evaluation, AD-60304, AD-60331, and AD-60344. The nucleotidesequences of the sense and antisense strands of these iRNA agents areprovided in Table 18. As indicated in Table 19, the nucleotide sequenceof AD-60304 is a perfect match to the mouse and rat nucleotidesequences. The nucleotide sequence of AD-60331 and the nucleotidesequence of AD-60344 have nucleotide mismatches (“MM”; see bolded,underlined nucleotides) to the mouse gene but have activity in mousehepatocytes.

C57BL/6 mice (N=3 per group) were injected subcutaneously with either 1mg/kg or 10 mg/kg of GalNAc conjugated duplexes or an equal volume of 1×Dulbecco's Phosphate-Buffered Saline (DPBS) (Life Technologies, Cat#14040133). Ninety-six hours later, mice were euthanized and the liverswere dissected and flash frozen in liquid nitrogen. Livers were groundin a 2000 Geno/Grinder (SPEX SamplePrep, Metuchen, N.J.). Approximately10 mg of liver powder per sample was used for RNA isolation. Sampleswere first homogenized in a TissueLyserII (Qiagen Inc, Valencia, Calif.)and then RNA was extracted using a RNeasy 96 Universal Tissue Kit(Qiagen Inc, Cat #74881) following manufacturer's protocol usingvacuum/spin technology. RNA concentration was measured by a NanoDrop8000 (Thermo Scientific, Wilmington, Del.) and was adjusted to 100ng/μl. cDNA was prepared and RT-PCR were performed as described above.

FIG. 2 demonstrates the efficacy of the CFB iRNAs to inhibit CFB mRNA ata dose of either 1 mg/kg or 10 mg/kg. At the 10 mg/kg dose, an averageof about 80% silencing was observed for all three iRNAs tested. At the 1mg/kg dose, an average of about 30% silencing was observed for AD-60331and AD-60344.

The ability of AD-60331 to suppress expression of CFB mRNA in vivo wasalso assessed using a single dose of 1.25 mg/kg, 2.5 mg/kg, and 10mg/kg. C57BL/6 mice were injected subcutaneously with the foregoingdoses and seventy hours later, mice were euthanized. RNA isolation formthe livers of the animals, cDNA preparation, and RT-PCR were performedas described above. FIG. 3 demonstrates that AD-60331 reduces CFB mRNAin a dose responsive manner, with an ED₅₀ of about 2.5 mg/kg. It isexpected that when introduced into human subjects, these iRNAs will beeven more effective given the design of the sequences.

TABLE 18 Sense Sequence Antisense Sequence(SEQ ID NOS 481-483, respectively, (SEQ ID NOS 484-486, respectively,Duplex in order of appearance) in order of appearance) speciesAD-60304.1 GfsasUfuGfaGfaAfGfGfuGfgCfgAfgUfuAfL96usAfsaCfuCfgCfcAfccuUfcUfcAfaUfcsasa MR AD-60331.1AfsgsCfaAfcAfuGfUfGfuUfcAfaAfgUfcAfL96usGfsaCfuUfuGfaAfcacAfuGfuUfgCfuscsa HC AD-60344.1GfscsUfgUfgGfuGfUfCfuGfaGfuAfcUfuUfL96asAfsaGfuAfcUfcAfgacAfcCfaCfaGfcscsc HC

TABLE 19 Antisense MM to mouse Antisense MM to rat (bold, underline)(bold, underline) (SEQ ID NOS 487-489, (SEQ ID NOS 490-492,respectively, respectively, PrimaryMouse Hep3b Duplexin order of appearance) in order of appearance) IC50(nM) IC50(nM)AD-60304.1 UAACUCGCCACCUUCUCAAUCAA UAACUCGCCACCUUCUCAAUCAA 0.028 2.876AD-60331.1 UGACUUU

AACACAUG

UGCUCA UGAC

UUGAACACAUG

UGCUCA 0.031 0.225 AD-60344.1

AAGUACUCAGACACCAC

GCCC

AAGUACUC

GACACCAC

GCCC 0.017 0.347

1-3. (canceled)
 4. The dsRNA agent of claim 21, wherein each strand isindependently 17-25 nucleotides in length.
 5. The dsRNA agent of claim21, wherein each strand is independently 17-23 nucleotides in length. 6.The dsRNA agent of claim 21, wherein each strand is independently 17-21nucleotides in length.
 7. The dsRNA agent of claim 21, whereinsubstantially all of the nucleotides of the sense strand andsubstantially all of the nucleotides of the antisense strand aremodified nucleotides.
 8. The dsRNA agent of claim 21, wherein all of thenucleotides of the sense strand and all of the nucleotides of theantisense strand are modified nucleotides.
 9. The dsRNA agent of claim21, wherein the at least one modified nucleotides has a nucleotidemodification selected from the group consisting of an LNA, a CRN, a cET,aUNA, an HNA, a CeNA, a 2′-methoxyethyl, a 2′-O-methyl, a 2′-O-alkyl, a2′-O-allyl, a 2′-C-allyl, a 2′-fluoro, a 2′-deoxy, a 2′-hydroxylmodification, and combinations thereof.
 10. The dsRNA agent of claim 21,wherein the at least one modified nucleotide is selected from the groupconsisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, anabasic nucleotide, and a 2′-amino modified nucleotide.
 11. The dsRNAagent of claim 21, wherein the at least one modified nucleotide is a2′-O-methyl modified nucleotide or a 2′-fluoro modified nucleotide. 12.The dsRNA agent of claim 21, wherein the ligand comprises anN-acetylgalactosamine (GalNAc) derivative.
 13. The dsRNA agent of claim21, wherein at least one of the 5′-end or the 3′-end of the sense strandof the dsRNA agent is a blunt end.
 14. (canceled)
 15. The dsRNA agent ofclaim 21, wherein the dsRNA agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage.
 16. ThedsRNA agent of claim 15, wherein the phosphorothioate ormethylphosphonate internucleotide linkage is at the 3′-terminus of onestrand.
 17. The dsRNA agent of claim 16, wherein the phosphorothioate ormethylphosphonate internucleotide linkage is at both the 5′- and3′-terminus of one strand.
 18. (canceled)
 19. An isolated cellcontaining the dsRNA agent of claim
 21. 20. A pharmaceutical compositionfor inhibiting expression of a complement component C3 gene, comprisingthe dsRNA agent of claim
 21. 21. A double stranded ribonucleic acid(dsRNA) agent selected from the group consisting of; (a) a dsRNA agentfor inhibiting expression of complement factor B (CFB) in a cell,wherein said dsRNA agent comprises a sense strand and an antisensestrand, wherein said sense strand comprises at least 15 contiguousnucleotides from any one of the nucleotide sequence of SEQ ID NOs:1-5,27, and 30, and said antisense strand comprises at least 15 contiguousnucleotides from any one of the nucleotide sequence of SEQ ID NOs:12-16,33, and 36; (b) a dsRNA agent for inhibiting expression of complementfactor B (CFB) in a cell, wherein said dsRNA comprises a sense strandand an antisense strand, the antisense strand comprising a region ofcomplementarity which comprises at least 15 contiguous nucleotides fromany one of the antisense sequences listed in Table 3 and 4; (c) a dsRNAagent for inhibiting expression of complement component C3 in a cell,wherein said dsRNA comprises a sense strand and an antisense strand, theantisense strand comprising a region of complementarity which comprisesat least 15 contiguous nucleotides from any one of the antisensesequences listed in Table 5 and 6; (d) a dsRNA agent for inhibitingexpression of complement component C3 in a cell, wherein said dsRNAagent comprises a sense strand and an antisense strand, wherein saidsense strand comprises at least 15 contiguous nucleotides from any oneof the nucleotide sequence of SEQ ID NOs:6-8, 28, and 31, and saidantisense strand comprises at least 15 contiguous nucleotides from anyone of the nucleotide sequence of SEQ ID NOs:17-19, 34, and 37; (e) adsRNA agent for inhibiting expression of complement component C9 in acell, wherein said dsRNA comprises a sense strand and an antisensestrand, wherein said sense strand comprises at least 15 contiguousnucleotides from any one of the nucleotide sequence of SEQ ID NOs:9-11,29, and 32, and said antisense strand comprises at least 15 contiguousnucleotides from any one of the nucleotide sequence of SEQ ID NOs:20-22,35, and 38; and (f) a dsRNA agent for inhibiting expression ofcomplement component C9 in a cell, wherein said dsRNA agent comprises asense strand and an antisense strand, the antisense strand comprising aregion of complementarity which comprises at least 15 contiguousnucleotides from any one of the antisense sequences listed in Table 7and 8; wherein the sense strand and the antisense strand form a doublestranded region, wherein the dsRNA agent comprises at least one modifiednucleotide, and a ligand.
 22. A method of inhibiting complement factor B(CFB) expression in a cell, the method comprising: (a) contacting thecell with the agent of claim 21; and (b) maintaining the cell producedin step (a) for a time sufficient to obtain degradation of the mRNAtranscript of a CFB gene, thereby inhibiting expression of the CFB genein the cell.
 23. A method of inhibiting complement component 3 (C3)expression in a cell, the method comprising: (a) contacting the cellwith the agent of claim 21; and (b) maintaining the cell produced instep (a) for a time sufficient to obtain degradation of the mRNAtranscript of a C3 gene, thereby inhibiting expression of the C3 gene inthe cell.
 24. A method of inhibiting complement component 9 (C9)expression in a cell, the method comprising: (a) contacting the cellwith the agent of claim 21; and (b) maintaining the cell produced instep (a) for a time sufficient to obtain degradation of the mRNAtranscript of a C9 gene, thereby inhibiting expression of the C9 gene inthe cell.